Article

FastT

rackSequential Duplications of an Ancient Member of theDnaJ-Family Expanded the Functional Chaperone Networkin the Eukaryotic CytosolChandan Sahi,y,z,1 Jacek Kominek,y,2 Thomas Ziegelhoffer,y,1 Hyun Young Yu,1 Maciej Baranowski,2

Jaroslaw Marszalek,*,1,2 and Elizabeth A. Craig*,1

1Department of Biochemistry, University of Wisconsin–Madison2Department of Molecular and Cellular Biology, Faculty of Biotechnology, University of Gdansk, Gdansk, PolandzPresent address: Department of Biology, Indian Institute of Science Education and Research Bhopal, Bhopal, Madhya Pradesh, IndiayThese authors contributed equally to this work.

*Corresponding authors: E-mail: ecraig@wisc.edu; marszalk@biotech.ug.gda.pl.

Associate editor: David Irwin

Abstract

Across eukaryotes, Hsp70-based chaperone machineries display an underlying unity in their sequence, structure, andbiochemical mechanism of action, while working in a myriad of cellular processes. In good part, this extraordinaryfunctional versatility is derived from the ability of a single Hsp70 to interact with an array of J-protein cochaperones toform a functional chaperone network. Among J-proteins, the DnaJ-type is the most prevalent, being present in all threekingdoms and in several different compartments of eukaryotic cells. However, because these ancient DnaJ-type proteinsdiverged at the base of the eukaryotic phylogeny, little is understood about the evolutionary basis of their diversificationand thus the functional expansion of the chaperone network. Here, we report results of evolutionary and experimentalanalyses of two more recent members of the cytosolic DnaJ family of Saccharomyces cerevisiae, Xdj1 and Apj1, whichemerged by sequential duplications of the ancient YDJ1 in Ascomycota. Sequence comparison and molecular modelingrevealed that both Xdj1 and Apj1 maintained a domain organization similar to that of multifunctional Ydj1. However,despite these similarities, both Xdj1 and Apj1 evolved highly specialized functions. Xdj1 plays a unique role in thetranslocation of proteins from the cytosol into mitochondria. Apj1’s specialized role is related to degradation of sumo-lyated proteins. Together these data provide the first clear example of cochaperone duplicates that evolved specializedfunctions, allowing expansion of the chaperone functional network, while maintaining the overall structural organizationof their parental gene.

Key words: J-protein, Hsp70, gene duplication, yeast, Hsp40, divergent evolution.

IntroductionAll Hsp70-based molecular chaperone machineries use thesame fundamental biochemical mechanism of action, cyclesof interaction with client proteins driven by ATP binding, andhydrolysis to function in many essential cellular processes(Bukau et al. 2006; Mayer 2010). These processes include fold-ing of nascent chains as they emerge from ribosomes, drivingprotein translocation across membranes, protecting cellsfrom heat stress, and facilitating biogenesis of Fe/S clusters(Kampinga and Craig 2010; Hartl et al. 2011). In good part, thisfunctional versatility is due to the fact that Hsp70s interactwith an array of J-protein cochaperones to form a functionalchaperone network (Craig et al. 2006). All members of theJ-protein superfamily possess a J-domain that binds Hsp70and is responsible for stimulation of Hsp70’s ATPase activity,an obligatory step for stabilizing Hsp70’s interaction withclient protein. However, outside their J-domains, J-proteinsvary widely in sequence and structure (Kampinga and Craig2010). Even the placement of the J-domain in their overall

topology can differ. Often these “extra-J-domain” regionsinteract with client proteins, targeting them to Hsp70.Thus, binding of a client to a J-protein prioritizes its interac-tion with Hsp70 (fig. 1). The cycle of interaction of clients withHsp70 initiated by its binding to a J-protein, is then followedby transfer to Hsp70, an interaction which is stabilized uponATP hydrolysis. The cycle culminates upon Hsp70’s interac-tion with a nucleotide exchange factor, which effects ADPrelease, resulting in exchange of ATP for ADP and dissociationof the client protein from Hsp70.

The first J-protein identified was DnaJ of Escherichia coli.The DnaJ family members (often referred to as Hsp40s) pro-liferated in eukaryotic cells with paralogous proteins presentin the endoplasmic reticulum, cytosol/nucleus, and mito-chondrium, where they function with compartment-specificHsp70 partners (Walsh et al. 2004; Kampinga and Craig 2010).Members of the DnaJ family, which are typically homodimers,are structurally complex. They have an approximately 70-residue N-terminal J-domain, followed by a region rich in

� The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, pleasee-mail: journals.permissions@oup.com

Mol. Biol. Evol. 30(5):985–998 doi:10.1093/molbev/mst008 Advance Access publication January 16, 2013 985

at University of W

isconsin Madison - G

eneral Library System

on May 20, 2013

http://mbe.oxfordjournals.org/

Dow

nloaded from

glycines and/or phenylalanines (G/F region) and a C-terminalregion that is critical for client–protein interaction. Structuralwork established that the C-terminal region of Ydj1, an abun-dant member of the DnaJ family in the cytosol of the buddingyeast Saccharomyces cerevisiae, is composed of two barreltopology domains, called CTD1 and CTD2, with CTD1having a Zinc finger-like region (ZnFR) extruding from it. Ahydrophobic client peptide-binding site was revealed in CTD1when the structure of a cocrystal of the C-terminus of Ydj1and a client peptide was solved (Li et al. 2003; Summers et al.2009). Single amino acid alterations within this cleft affectedthe ability of Ydj1 to bind and facilitate refolding of clientproteins such as denatured luciferase (Li and Sha 2005). TheZnFR has been suggested to play roles in client protein bind-ing and/or interaction with Hsp70 (Linke et al. 2003; Fan et al.2005).

The cytosol/nucleus of the budding yeast, S. cerevisiae,which contains a large functional network of 13 J-proteinsuperfamily members, has been useful as a model systemfor understanding the diversity of J-protein function in cellularprocesses. This has particularly been the case for J-proteinsstructurally different from those of the DnaJ-type, such asCwc23 (Sahi et al. 2010) and Jjj1 (Demoinet et al. 2007;Meyer et al. 2007), which are involved in mRNA splicingand ribosome biogenesis, respectively. The abundantDnaJ-type J-protein of the cytosol/nucleus, Ydj1, has alsobeen studied extensively and shown to play general roles inde novo protein folding, protein translocation across mem-branes, and thermotolerance (Becker et al. 1996; Glover andLindquist 1998; Summers et al. 2009). However, little informa-tion is available regarding the evolutionary origin and possiblefunctional diversification and specialization of DnaJ-typeJ-proteins in any eukaryotic compartment. Here, we reportthe identification and analysis of two additional DnaJ-typeproteins of the S. cerevisiae cytosol, Xdj1 and Apj1. The resultsof our evolutionary analysis are consistent with the idea thatboth proteins evolved by sequential duplications of the geneencoding Ydj1; Xdj1 in a common ancestor of Ascomycota,Apj1 more recently in a common ancestor of theSaccharomycetaceae and CTG clade (Dujon 2010) to which

both S. cerevisiae and Candida albicans belong. Sequenceanalyses revealed that, consistent with their low abundance,both Xdj1 and Apj1 evolve significantly faster than Ydj1, andthat their presence did not affect the mode or the rate of Ydj1evolution. But, sequence analysis alone was not sufficient topredict whether Xdj1 and Apj1 had diversified functionallyfrom Ydj1. However, our experimental work revealed thatboth Xdj1 and Apj1 have specialized roles, with Xdj1 beinginvolved in mitochondrial protein import and Apj1 playing arole in degradation of sumolyated proteins.

Results

Three Members of DnaJ-Like Subfamily ofS. cerevisiae Cytosol

Like Ydj1, two other J-proteins of the cytosol, Apj1 and Xdj1,have the easily discernible characteristics that historicallyplaced them in a group called class I J-proteins (Cheethamand Caplan 1998): an N-terminal J-domain, an adjacent regionrich in glycine and/or phenylalanine residues (G/F region) andfour cysteine residues appropriately spaced to form a Znfinger (fig. 2A). To determine whether Xdj1 and Apj1 couldbe considered of the DnaJ-type (e.g., having the beta-barreltopology of CTD1 and CTD2), we extended our comparisonusing molecular modeling of the sequences C-terminal to theG/F region. Apj1 and Xdj1 had predicted topologies very sim-ilar to CTD1 and CTD2 of Ydj1 (fig. 2A). The Ydj1client-binding site, revealed by cocrystallization with a clientpeptide (Li et al. 2003), is composed of six hydrophobic res-idues. Examination of the putative peptide binding clefts ofXdj1 and Apj1 revealed hydrophobic residues positioned sim-ilarly to those in the cleft of Ydj1 (fig. 2A). We conclude thatthe Ydj1, Apj1, and Xdj1 are members of the DnaJ family.However, we also found several differences amongst thethree proteins. First, although the three hydrophobic-bindingclefts were similarly positioned, the residues were not identi-cal (fig. 2A). Second, although Xdj1 has a predicted prenyla-tion site at its extreme C-terminus, similarly positioned tothat of the experimentally determined Ydj1 site (supplemen-tary fig. S1, Supplementary Material online), Apj1 does notcontain a C-terminal cysteine, a requirement for this post-translational modification. Third, the putative C-terminal di-merization region of Apj1 is longer than that of either Xdj1 orYdj1. Fourth, the G/F-rich region of Apj1 is almost 2-foldlonger than that of either Ydj1 or Xdj1, and although beingrich in G/F residues as required of class I J-proteins, it also hasserine, glutamine, and asparagine present in higher than ex-pected proportions (supplementary fig. S1, SupplementaryMaterial online). Herein, we refer to this segment of Apj1G/F-S region.

As our goal was to better understand both the functionaland evolutionary relationships among these three J-proteins,we carried out three experimental comparisons. First, toassess their cellular abundance, we determined their levelsin whole cell extracts derived from wild-type (WT) cells,using purified proteins as standards and antibodies specificfor each protein. Our results indicate that WT cells haveapproximately 90,000 Ydj1 molecules/cell (supplementary

FIG. 1. J-protein dependent cycle of interaction of Hsp70 with clientprotein. J-protein (light gray) binds client protein (line). Client protein isdelivered to ATP-bound Hsp70, which has a fast on and off rate forclient. J-domain of J-protein (J) stimulates Hsp70’s ATPase activity. Asthe resulting ADP-bound Hsp70 (dark gray) has a slow client off rate, theinteraction of the client with Hsp70 is stabilized. Interaction of a nucle-otide exchange factor (NEF) facilitates release of ADP, followed by bind-ing of ATP, causing client dissociation and priming of Hsp70 for anothercycle of interaction.

986

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

fig. S2, Supplementary Material online). Xdj1 and Apj1 aresignificantly less abundant, at 500 and 100 molecules/cell,respectively. Second, we tested side-by-side the affect of de-letion of the individual genes on cell growth. Consistent withprevious reports (Caplan and Douglas 1991; Caplan et al.1992), the absence of Ydj1 resulted in severe growth defects(fig. 2B). However, no obvious growth defects were observedfor apj1� or xdj1� under a variety of conditions. Third, weset up systems to purify the three proteins to test their abilityto stimulate Hsp70’s ATPase activity. Xdj1 and Ydj1 werereadily purified, whereas Apj1 was prone to aggregation.However, sufficient quantities of Apj1 were obtained toassess the ability to stimulate the ATPase activity of Ssa1,the abundant cytosolic/nuclear Hsp70. Like previously ob-served for Ydj1, Xdj1, and Apj1 stimulated Ssa1 efficiently

(fig. 2C). We conclude that Ydj1, Apj1, and Xdj1 are membersof DnaJ-like family and are able to function with the Ssa classof Hsp70 in the cytosol/nucleus of S. cerevisiae.

Ydj1 and Xdj1 Are Found throughout theAscomycetes Species

To begin to understand the evolutionary relationshipsamongst cytosolic DnaJ-like proteins, we first focused onXdj1 because, as described earlier, some features of Xdj1 aremore similar to those of Ydj1 (i.e., the size and amino acidcomposition of the G/F region and the putative prenylationsite) than are those of Apj1. We searched for Ydj1 and Xdj1orthologs in 43 Ascomycetes species (supplementary fig. S3,Supplementary Material online) using the S. cerevisiae

FIG. 2. Domain structures, deletion phenotypes, and cochaperone activities of DnaJ-like proteins from the cytosol/nucleolus of Saccharomycescerevisiae. (A) (Top) Diagrams indicating domain structure of DnaJ-like proteins, drawn to scale: J-domain (J), glycine/phenylalanine-rich region(GF), beta barrel domain I with ZnFR (CTDI + ZnF), beta barrel domain II (CTDII), dimerization domain (DD). Amino acid residues indicated aboveeach diagram indicate the boundaries of the regions. (Middle) Ribbon diagrams of CTDI/II region. Ydj1-CTDI CTDII domains cocrystallized with clientpeptide (magenta) (PDB:1NLT); Xdj1 and Apj1 homology models prepared using SWISS-MODEL (Kiefer et al. 2009) and the Ydj1 structure as atemplate. (Bottom) Space-filled diagrams of peptide binding cleft. Ydj1, based on crystal structure, with client peptide shown in magenta and Ydj1residues involved in peptide binding shown in color, as indicated in the sequence alignment (bottom). Homology models of peptide-binding cleft ofXdj1 and Apj1. (B) WT and mutant cells, as indicated, were plated as 10-fold dilutions on rich media and incubated at indicated temperatures for 3 days.(C) Ssa1 ATPase activity under single-turnover conditions was measured in the presence of Apj1, Xdj1, and Ydj1 as indicated, or without addition(none). (Left) Hydrolysis of bound ATP over time (average of two determinations); (right) fold-change in the apparent first order rate constant for ATPhydrolysis determined using the data from left panel.

987

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

sequences as a query for BLAST analysis. All surveyed speciesexhibited two significant BLAST hits (fig. 3), which we couldreadily identify as orthologs of Ydj1 and Xdj1. ReciprocalBLAST confirmed our predictions.

Next, we asked whether Ydj1 and Xdj1 orthologs were alsopresent in well-annotated genomes of other fungi, metazoa,plantae, and protozoa. The BLAST search using the sequenceof S. cerevisiae YDJ1 and XDJ1 as query provided one or morehits in each of the 32 surveyed species (supplementary fig. S4,Supplementary Material online). However, in each case, theYDJ1 hits had higher scores than those of XDJ1 hits. Moreover,except for 12 cases, reciprocal BLAST of the top three putativeXDJ1 orthologs returned YDJ1 as a best hit in S. cerevisiae.Of the 12 questionable cases, none returned XDJ1, but ratherother S. cerevisiae J-proteins (SCJ1, JEM1, and SIS1). That eventhe best XDJ1 hits are more similar to YDJ1 than to XDJ1supports the hypothesis that XDJ1 orthologs are not presentin the surveyed species. Thus, the birth of XDJ1 can be tracedto the common ancestor of the Ascomycota clade. In

contrast, orthologs of YDJ1 were found in all surveyed eukary-otic genomes, consistent with the hypothesis that this ancientmember of DnaJ-like subfamily is indispensable for properfunctioning of the cytosol/nucleus.

Xdj1 Toxicity, Whose Peptide-Binding SpecificityDiffers from Ydj1, Is Relieved by Reduction inPeptide Binding

The maintenance of Xdj1 throughout Ascomycetes species,even though expressed at levels almost 200-fold less thanYdj1, points to Xdj1 having a specialized function. To under-stand what biological role Xdj1 plays, we began an analysis ofthe effects of both its absence and its overexpression. Wefound that increased expression of Xdj1 was detrimental.No transformants with plasmids having XDJ1 under the con-trol of either the strong TEF or GPD promoter were recovered.However, transformants expressing Xdj1 under the controlof the weaker ADH1 promoter were obtained. These cells,which expressed Xdj1 at levels approximately four timeshigher than that found in WT cells, grew more poorly(fig. 4A). To determine whether this toxicity was dependenton its Hsp70 cochaperone function (e.g., as a J-protein), weused a mutant encoding an H to Q alteration in the invariantHPD motif of the J-domain, which has been shown in anumber of J-proteins to inactivate the functional interactionof this domain with its Hsp70 partner (Kampinga and Craig2010). Cells expressing Xdj1H37Q from the ADH promotergrew as well as WT cells. From this observation, we concludedthat toxicity was dependent on Xdj1’s cochaperone functionwith Hsp70 (fig. 4B). Next, we tested variants having alter-ations in one of five residues in the putative peptide-bindingcleft (fig. 2). Alanine alterations at L151, I243, L275, and F277alleviated toxicity, whereas F153A did not (fig. 4C).

The results described earlier indicate that the toxicitycaused by Xdj1 overexpression requires both interactionwith Hsp70 and binding of client protein, suggesting to usthat “inappropriate” or prolonged association of certain clientproteins with Hsp70 due to targeting by Xdj1 is the cause ofthe toxicity. To test this idea, we overexpressed Sse1, a nucle-otide exchange factor for the Hsp70 Ssa (Dragovic et al. 2006;Raviol et al. 2006), concurrently with overexpression of Xdj1.The logic behind this test was that release of ADP from theHsp70 Ssa by the action of Sse would speed up the cycleof interaction of Hsp70 with client protein. In this way, theresidency time of client proteins targeted by Xdj1 would bereduced. We found that, as hypothesized, overexpression ofSse1 partially relieved the toxicity of Xdj1 overexpression, ascells having levels of Xdj1 4-fold above normal grew betterwhen Sse1 expression was higher than that normally found(fig. 4D).

An increase of Xdj1 levels to only 2,000 molecules/cell re-sulted in toxicity, whereas Ydj1 is normally present at 90,000molecules/cell, suggested to us that Xdj1 and Ydj1 may havedifferent peptide-binding specificities. To test this idea, weproceeded to search for peptides that bound to Xdj1 orYdj1. Using purified Xdj1 or Ydj1, we screened a phage libraryexpressing on their surface random 12 residue peptides.

FIG. 3. Phylogenetic distributions of YDJ1, XDJ1, and APJ1. The phylo-genetic distributions of genes are presented in a simplified cladogram ofthe fungal phylum Ascomycota, which consists of three subphylaTaphrinomycotina, Pezizomycotina, and Saccharomycotina (Dujon2010). The species of the Taphrinomycotina and Pezizomycotinaclades are represented as separate triangles. The Saccharomycotinaspecies were separated into three clades: Yarrowia, CTG, andSaccharomycetaceae. The Saccharomycetaceae consisting primary ofspecies from the genera Saccharomyces and Kluyveromyces was furtherdivided into the lineage that underwent the whole genome duplication(post-WGD) and the one that did not (pre-WGD). Shading in thepost-WGD triangle indicates five closely relate species belonging toSaccharomyces sensu stricto. One representative species of each cladeis named for clarity, with the total number of surveyed species indicatedin parentheses. The presence (+) or absence (�) of the orthologs isindicated. The phylogenetic position of the putative origin of Xdj1 ismarked with an open circle and those of Apj1 with a closed circle. Forthe list of additionally surveyed eukaryotic species, see supplementaryfigure S4, Supplementary Material online.

988

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

We obtained phage from the Xdj1 and Ydj1 screen having4 and 10 distinct peptide sequences, respectively (supplemen-tary fig. S5, Supplementary Material online). To assesswhether these peptides were indeed interacting in theclient-binding cleft of the subdomain CTD1 (fig. 2A), we com-pared phage interaction of WT proteins and variants havingalterations in the cleft, Xdj1L275S and Ydj1L135A. Two of theXdj1- and 9 of the Ydj1-selected phage showed substantialreduction in binding to the variants compared with WT pro-tein (fig. 4E). Next, we “cross-tested” the 11 phage, comparingtheir ability to bind the J-protein not used for their selectionwith the J-protein used in the initial screening. In all cases, thephage bound more efficiently to the protein used for theirselection than to the other J-protein (fig. 4F). The bestcross-binder, Y6, only bound Xdj1 18% as efficiently underthe conditions used for screening than it bound Ydj1, withwhich it was selected. Together our results are consistent withthe conclusion that the peptide-binding specificities of Xdj1and Ydj1 are obviously distinct and that a decrease in theaffinity of Xdj1 for client protein reduces its toxicity.

Xdj1 Functions in Mitochondrial Import

Toxicity is a useful tool for understanding Xdj1. However, itsanalysis did not reveal information regarding Xdj1’s biological

function. As we had observed no obvious growth phenotypesof xdj1� cells, we set up tests to validate synthetic geneticinteractions previously noted in genomic wide screens. Themost dramatic report was a lethal interaction with a temper-ature sensitive allele of the molecular chaperone Hsp90 (Zhaoet al. 2005). However, we did not detect any genetic interac-tion (supplementary fig. S6, Supplementary Material online).We also noted the report in another genomic-wide screen(Costanzo et al. 2010) of a genetic interaction, between dele-tions of XDJ1 and PAM17, which encodes a nonessential com-ponent of the protein import apparatus of the mitochondrialinner membrane (van der Laan et al. 2005). As Xdj1, but notYdj1, had been found in a proteomic analysis of mitochon-drial outer membranes (Burri et al. 2006), we pursued thisobservation. Although pam17� cells, such as xdj1� cells hadno obvious effect on cell growth, xdj1� pam17� cells grewsomewhat more poorly than WT cells at 18 �C (fig. 5A). Thissynthetic growth interaction was dependent on a functionalinteraction with Hsp70, as xdj1H37Q pam17� cells also grewpoorly at 18 �C (fig. 5B). Many proteins synthesized on cyto-solic ribosomes, but destined for mitochondria, contain anN-terminal-targeting presequence that is cleaved after trans-location to the matrix. Thus, a defect in mitochondrial importcan be detected by monitoring the accumulation of the

FIG. 4. Moderate over-expression of Xdj1 is toxic: (A) Lower panel. Ten-fold serial dilutions of WT cells transformed with empty vector (—) or pRS414-ADH-Xdj1 (ADH-Xdj1) were spotted on Trp omission media and incubated at 30 �C for 2 days. (Upper panel) Total protein isolated from xdj1� cellstransformed with empty vector (xdj1�) or WT cells transformed with either empty vector (—) or pRS414-ADH-Xdj1 (ADH-Xdj1) was resolved byelectrophoresis, electro-blotted, and probed with anti-Xdj1 (Xdj1) and, as loading control, anti-Ssc1 antibody (c). (B) Ten-fold serial dilutions of xdj1�

cells transformed with empty vector (—), pRS414-ADH-Xdj1 (WT) or pRS414-ADH-Xdj1H37Q (H37Q) were spotted on Trp omission medium andincubated at 30 �C for 2 days. (C) Ten-fold serial dilution of xdj1� cells transformed with pRS414-ADH-Xdj1 (WT) or pRS414-ADH-Xdj1 encoding theindicated variants having single amino acid alterations in the putative peptide-binding cleft were spotted on Trp omission medium and incubated at30 �C for 2 days. (D) Ten-fold serial dilutions of xdj1� cells transformed with empty vector (—, left) or pRS413-GPD-Sse1 (GPD-Sse1, right) and a secondplasmid: empty (—), pRS414-ADH-Xdj1 (WT), pRS414-ADH-Xdj1H37Q (H37Q), or pRS414-ADH-Xdj1L275A (L275A) were spotted on Trp-His doubleomission plates and incubated at 30 �C for 2 days. (E, F) Purified Xdj1, Xdj1L275A, Ydj1, or Ydj1L135A were immobilized on 96-well plates and assessed fortheir ability to bind coliphage expressing on their surface various 12-mer peptides (4� 107 phage/well) that were selected using Xdj1 (left) or Ydj1(right) using phage-specific antibody. (E) 4 and 12 unique coliphage selected for binding to Xdj1 (left) or Ydj1 (right) in panning experiments were testedfor binding to Xdj1 and Xdj1L275A or Ydj1 and Ydj1L135A, respectively. (F) Indicated coliphage whose binding to cognate J protein was significantlyimpaired in cleft mutants were tested for specificity of binding to either Xdj1 or Ydj1.

989

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

precursor form of mitochondrial proteins, including Mdj1(Pais et al. 2011). A small amount of Mdj1 precursor accu-mulated in pam17� cells at both 23�C and 18 �C (fig. 5C).This effect was exacerbated when Xdj1 was also absent, val-idating the synthetic genetic growth interaction between adeletion of XDJ1 and a deletion of PAM17. We also testedwhether precursor accumulated in cells overexpressing Xdj1.Approximately one-third of the Mdj1 protein was present inthe precursor form in cells expressing Xdj1 at levels 4-foldabove normal (fig. 5D). This accumulation was amelioratedby alterations in Xdj1 that affect J-domain function (H37Q) orclient protein binding (L275A). Together our in vivo resultsindicate that Xdj1 specializes in facilitating import of proteinsfrom the cytosol into mitochondria, and that the cellulartoxicity caused by modest overexpression of Xdj1 is due toaffects on the import process, likely by increased associationof client proteins to Hsp70, that are targeted by Xdj1.

APJ1 Arose via Duplication of YDJ1

Next, turning our attention to Apj1, we used BLAST anal-ysis to search for the presence of APJ1 orthologs inAscomycetes species, using the S. cerevisiae sequence as aquery. Using reciprocal BLAST, we could confidently iden-tify the candidate sequence as an APJ1 ortholog withinspecies belonging to the Saccharomycetaceae. However,

the same query in the sister CTG clade returned threehits with similar E values for each analyzed species, twoof which we had previously identified by means of recip-rocal BLAST search, phylogeny (discussed later) and thepresence of the C-terminal prenylation motif (CXXQ) asorthologs of YDJ1 and XDJ1. More in-depth analysis of thedomain structure and presence of conserved sequencemotifs such as a longer and an S-, Q-, and N-rich G/F-Sregion, as well as protein phylogeny, allowed us to annotatethe remaining hits from the CTG clade as putative APJ1orthologs. When the sequence of the putative APJ1 ortho-log from Candida albicans, a member of the CTG clade,was used as reciprocal BLAST query within the CTG clade,best results corresponded to the annotated APJ1 sequences.However, when a similar analysis was carried using ortho-logs from species belonging to the Saccharomycetaceae,similar E values were returned for previously identifiedAPJ1, YDJ1, and XDJ1 (data not shown), indicating thatAPJ1 orthologs exhibited a high level of clade-specific se-quence divergence.

Outside the Saccharomycetaceae and CTG clade, onlytwo high score BLAST hits were obtained for each species,which we identified as orthologs of YDJ1 and XDJ1 (fig. 3).Moreover, reciprocal BLAST analysis confirmed the anno-tation of these two sequences in each genome tested.No Apj1 ortholog was found in species outside theSaccharomycetaceae and CTG clade. This phylogenetic dis-tribution of APJ1 exclusively in species forming a mono-phyletic group suggested to us that APJ1 arose in acommon ancestor of the Saccharomycetaceae and theCTG clade, most likely via a gene duplication event. Toidentify the parental APJ1 gene, we analyzed phylogeneticrelationships among APJ1, YDJ1, and XDJ1, using as an outgroup two sequences encoding DnaJ-like proteins function-ing in other cellular compartments: SCJ1 of the endoplas-mic reticulum and MDJ1 of the mitochondrium. Withinthis large maximum likelihood (ML) reconstruction of theamino acid–based phylogeny, the branching order of indi-vidual gene trees recapitulated the accepted species tree(Fitzpatrick et al. 2006) with the main phylogenetic groupsretrieved with high support (fig. 6 and supplementary fig.S7, Supplementary Material online). However, placement ofindividual species within the major groups was somewhatvariable, although no significant deviations from the ac-cepted taxonomy were observed. The discrepancies canlikely be explained by loss of the protein-specific phyloge-netic signal in the alignment of all five DnaJ-like proteinsused for tree inference. Inspection of the protein phylogenyindicated that APJ1, YDJ1, and XDJ1 formed a strongly sup-ported monophyletic clade, suggesting that DnaJ-like pro-teins present in the cytosol/nucleus are closely related(fig. 6 and supplementary fig. S7, Supplementary Materialonline). Moreover, APJ1 sequences formed a sister clade tothe YDJ1 sequences, indicating that APJ1 is most likely re-lated to YDJ1 by a gene duplication event that took placein a progenitor of the Saccharomycetaceae and the CTGclade.

FIG. 5. Xdj1 has a role in mitochondrial protein import. (A) Ten-foldserial dilutions of WT, xdj1�, pam17�, and xdj1� pam17� cells werespotted on rich media and incubated at 18 �C for 7 days. (B) Ten-foldserial dilutions of xdj1� pam17� cells transformed with empty vectorpRS313 (—), pRS313-Xdj1 (WT) or pRS313-Xdj1H37Q (H37Q) were spot-ted on a His omission plate and incubated at 18 �C for 7 days. (C, D)Total protein isolated from indicated strains were resolved by electro-phoresis, electro-blotted and probed with anti-Mdj1 and, as loadingcontrol, anti-Sse1 antibody (c). Precursor (p) and mature (m) formsof Mdj1 are indicated. (C) WT, xdj1�, pam17�, and xdj1� pam17�

cells grown at either 23 �C or 18 �C. (D) xdj1� cells expressing emptyvector pRS414 (—), pRS414-ADH-Xdj1 (WT), pRS414-ADH-Xdj1H37Q

(H37Q), pRS414-ADH-Xdj1L153A (L153A), and pRS414-ADH-Xdj1L275A

(L275A) grown at 30 �C. All samples were run on the same gel;dotted lines indicate splicing out of lanes having irrelevant samples.

990

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

Apj1’s Specialized Function Is Related to Sumo-Targeted Protein Degradation

APJ1’s retention in all postduplication species combined withits low expression levels in S. cerevisiae suggests that it hasfunctionally diverged from its parental gene. To gain a betterunderstanding of the functional differences between Apj1and Ydj1, we searched for a phenotype of apj1� cells. Aswe observed no growth defect of apj1� cells under a varietyof conditions, we extended our analysis to include syntheticgenetic interactions. Noting apj1� listed amongst candidatesin a global screen for synthetic genetic interactions with adeletion of SLX5, which encodes a subunit of a SUMO-targeted ubiquitin ligase (Pan et al. 2006), we compared thegrowth of slx5 and slx5� apj1� cells (fig. 7A). Although slx5�cells grew more slowly than WT cells at all temperaturestested, slx5� apj1� cells grew even more poorly and wereunable to form individual colonies at 37 �C. To verify thegenetic interaction between SLX5 and APJ1, we comparedaccumulation of SUMO-modified proteins by immunoblotanalysis, using SUMO-specific antibodies. As expected(Mullen and Brill 2008), a wide range of proteins having

SUMO modifications was observed in WT cells, with slx5�cells having higher levels. This greater amount of sumolyatedproteins in the absence of Slx5 is attributed to impairment ofthe ubiquitination of these proteins and thus their degrada-tion (fig. 7B). The level of sumolyated proteins in the slx5�apj1� double mutant was higher than that in the slx5�single mutant, consistent with Apj1 playing a role in the pro-teolysis of proteins targeted for degradation by sumoylation,and validating the genetic interaction between a deletion ofAPJ1 and a deletion of SLX5. To test whether the syntheticgenetic interaction was dependent on the ability of Apj1 tofunction as a J-protein, we made use of an H to Q alteration inthe J-domain, analogous to the Xdj1 variant described earlier.apj1H34Q was not able to substitute for WT APJ1 to allowgrowth at 37 �C (fig. 7C), indicating that Apj1 functions inprotein degradation as part of a larger Hsp70-based chaper-one machinery.

We took a genetic approach to define regions of Apj1critical for its specialized function. To assess possible roles ofthe G/F-S region and ZnFR in functional specificity, we cre-ated internal deletion mutations removing DNA segmentsencoding these regions. We were particularly interested inApj1’s G/F-S region. However, apj1�G/F-S slx5� consistentlygrew nearly as well as slx5� cells, even at 37 �C, indicatingthat the G/F-S region, though distinct from the G/F region ofYdj1, is not critical for function. On the other hand, deletionof the Zn-binding region had deleterious effects on Apj1 func-tion, as apj1�ZnFR slx5� cells grew nearly as poorly as apj1�slx5�. However, substitution of Apj1’s ZnFR with Ydj1’s re-stored function, indicating that the while the ZnFR is critical,it does not provide functional specificity for Apj1. These re-sults suggest that the specificity for Apj1 function lies else-where in the C-terminal segment. To address whetherpeptide binding is critical for function, six variants, eachhaving one of the hydrophobic cleft residues changed to aserine, were expressed in apj1� slx5� cells, and their ability tosubstitute for WT Apj1 tested. Three (I179S, L200S, andL277S) were unable to substitute for WT Apj1, as indicatedby failure of the double mutants to form colonies at 37 �C.Therefore, we concluded that client binding ability is criticalfor Apj1’s function in protein degradation.

Apj1 and Xdj1 Are Rapidly Evolving

Evident functional differences among the three cytosolicmembers of DnaJ-subfamily prompted us to compare therates of evolution of their sequences. Pair-wise comparisonsof all Ydj1 amino acid sequences revealed a high level ofconservation (�60% identity), whereas the sequence conser-vation was much lower for Xdj1 (�38% identity) and Apj1(�34% identity) (table 1). Consistent with their high se-quence variability, the branches of both the amino acid–based tree (fig. 6) and the nucleotide-based tree (data notshown) are longer for XDJ1 and APJ1 compared with YDJ1.Furthermore, analysis of codon evolution indicated thatnonsynonymous rates of evolution are significantly higherfor XDJ1 and APJ1 (dN = 0.08 ± 0.07 and 0.1 ± 0.09, respec-tively) than for YDJ1 (dN = 0.04 ± 0.03) (table 1). We also

FIG. 6. Phylogeny of Ydj1, Xdj1, and Apj1. ML-inferred phylogeny ofamino acid sequences of the Ydj1, Xdj1, and Apj1, rooted using Scj1and Mdj1 sequences from the set of surveyed fungi species. Scale is insubstitutions per amino acid site. Bootstrap support values out of 100pseudoreplicates are indicated. The complete phylogenetic tree, includ-ing fully resolved Scj1 and Mdj1 clades is provided as supplementaryfigure S7, Supplementary Material online.

991

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

noticed that the dN values determined only for YDJ1sequences from the species in which YDJ1 coexists withAPJ1 (YDJ1post-Apj1) or only for the YDJ1 sequences from spe-cies lacking APJ1 (YDJ1pre-APJ1) were identical, indicating thatthe coexistence of APJ1 with YDJ1 had no effect on the rate ofnonsynonymous substitutions in YDJ1.

As the per site frequency of synonymous substitutions wassaturated (dS> 1) for each analyzed gene, preventing us fromaccurate estimation of the dN/dS ratio, we proceeded toanalyze amino acid sequence evolution further. We searchedfor two types of divergence (Gu 1999, 2001): type I, sites highlyconserved in one group of paralogous sequences that arehighly variable in another group, and often interpreted asindication of relaxation of functional constraints and site-specific shift of evolutionary rate; and type II, sites conservedwithin each group of paralogs, but differ radically in theirbiochemical properties, and often interpreted as associatedwith putative functional changes. Using the DIVERGE soft-ware (Gu and Vander Velden 2002) to compare all Xdj1 to all

Ydj1 sequences, we obtained a moderately high coefficient oftype I sequence divergence �I (0.48 ± 0.05) (table 2). A similar�I value (0.39 ± 0.06) was obtained from comparison ofApj1 sequences with Ydj1post-Apj1. However, a lower �I

value (0.26 ± 0.05) was obtained from comparison betweenYdj1post-Apj1 and Ydj1pre-Apj1, suggesting that most of the typeI divergence was associated with Xdj1 and Apj1 sequences. Tofurther verify this prediction, we inspected multisequencealignments, searching for sites in strict agreement withthe type I divergence definition—invariant in one group ofcompared sequences but variable in another group. TheXdj1:Ydj1 comparison revealed 50 type I sites, of which48 were variable in the Xdj1 sequence (table 2). In the caseof the Apj1:Ydj1postApj1 comparison, we found 69 type I sites,with 67 of them being variable in Apj1. In striking contrast,the Ydj1postApj1: Ydj1preApj1 comparison identified 56 type Isites, but variable sites were distributed equally, 34 and 22,respectively, between two groups of sequences. We concludethat both Xdj1 and Apj1 exhibit elevated rates of sequence

FIG. 7. apj1� and slx5� have a synthetic genetic interaction related to Sumo-mediated degradation. (A) Ten-fold serial dilutions of WT, apj1�, slx5�,and apj1� slx5� cells were spotted on rich media and incubated at indicated temperatures for 2 days. All strains in upper and lower panels werespotted on the same plate; dotted line indicates that irrelevant strains were spliced out of the figure. (B) Extracts of total protein isolated from WT,apj1�, slx5�, or apj1� slx5� cells were resolved by electrophoresis, electroblotted and probed with antibodies specific for Smt3, Saccharomycescerevisiae sumo protein and, as a control, anti-Ssc1 antibodies (c). (C, D, E) Ten-fold serial dilutions of apj1� slx5� carrying the plasmids indicated belowwere spotted on media lacking tryptophan and incubated at indicated temperatures for 2 days. (C) pRS314-Apj1 (WT) or pRS314-Apj1H34Q (H34Q).(D) Empty vector (—), pRS314-Apj1 (WT), pRS314-Apj1�79-161 (�GFS), pRS314-Apj1�206-262 (�Zn) or pRS314-Apj1Ydj1-143-206 (Zn swap). (E)pRS314-Apj1 (WT), pRS314-Apj1I179S (I179S), pRS314-Apj1L198S (L198S), pRS314-Apj1L200S (L200S), pRS314-Apj1L277S (L277S), pRS314-Apj1V316S

(V316S), pRS314-Apj1I318S (I318S).

Table 1. Summary of Sequence Analysis.

Gene Sequence Statistics Codon Usage Bias Evolutionary Rates

Lengtha Identityb Similarityc ENC CAI dSall dNall dNpre-Apj1 dNpost-Apj1

YDJ1 411 ± 6 59.53 ± 11.7 73.18 ± 9.14 41.27 ± 5.81 0.36 ± 0.05 1.18 ± 1.04 0.04 ± 0.03 0.04 ± 0.04 0.04 ± 0.04

XDJ1 436 ± 22 37.78 ± 12.4* 55.26 ± 10.73* 50.89 ± 6.25* 0.13 ± 0.01* 1.58 ± 1.35 0.08 ± 0.07* 0.08 ± 0.08* 0.09 ± 0.08*

APJ1 513 ± 38 34.30 ± 15.2* 49.27 ± 14.54* 52.68 ± 4.62* 0.14 ± 0.01* 2.51 ± 2.34* 0.10 ± 0.09* — —

NOTE.—All values are shown as mean ± SD.aMean number of residues in the amino acid sequence.bMean pairwise amino acid sequence identity.cMean pairwise amino acid sequence similarity (see Materials and Methods for details).

*Significantly different from YDJ1 at the P = 0.01 level.

992

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

divergence, whereas that of Ydj1 was rather limited.Moreover, the divergence of Ydj1 was not affected by theemergence of Apj1 and thus, the coexistence of these twoproteins. However, in all cases, type I sites were distributedacross the entire alignment. Therefore, we were unable toidentify specific domains as being responsible for the increaseof the sequence divergence; rather the entire sequences ofboth Xdj1 and Apj1 were more variable than those of Ydj1(data not shown). Interestingly, despite relatively high level oftype I sequence divergence, we did not identify any type IIdivergent sites in any of the sequence comparisons discussedearlier.

The results of our analyses of YDJ1, XDJ1, and APJ1 se-quences described earlier are consistent with a scenario inwhich over a long evolutionary time scale both XDJ1 andAPJ1 evolved faster than YDJ1. To test whether this wasalso the case for a shorter time evolution scale, we analyzedcodon evolution for a set of five species that are closelyrelated to S. cerevisiae, belonging to the Saccharomycessensu stricto (Dujon 2010). As for this data set, the dSvalues were not saturated (dS� 1, table 3), and wecould analyze distribution of dN/dS ratio along the phylog-eny. Specifically, for each set of orthologous sequences(XDJ1, APJ1, and YDJ1), we evaluated the hypothesis thatall branches of the phylogeny exhibit the same dN/dS ratioagainst a set of hypotheses under which the dN/dS ratioalong the external branch for a given species was differentfrom that in the rest of the phylogeny. All the analyzedgenes did not reject the null hypothesis of a uniform dN/dSratio across the phylogeny at the P = 0.01 level. However, asexpected, YDJ1 evolved more slowly than either XDJ1 orAPJ1 (dN/dS values of 0.027, 0.155, and 0.080, respectively)(table 3). Finally, as the dN/dS values obtained for eachanalyzed gene were obviously lower than 1, we concludedthat on a finer time scale YDJ1, XDJ1, and APJ1 are subjectto strong purifying selection.

DiscussionThe results presented here support the idea that the DnaJtype J-proteins of the S. cerevisiae cytosol, Apj1 and Xdj1, playspecialized roles in critical biological functions—proteolysisand protein translocation across membranes, respectively.The absence of Apj1 exacerbated the defect in degradationof sumolyated proteins when the cell was challenged by areduction in sumo-dependent ubiquitin ligase activity.Translocation of proteins into mitochondria was defectivewhen xdj1� cells were challenged by the absence of a com-ponent of the inner membrane protein translocase. Thatthese phenotypes were observed is remarkable given thatYdj1, the third DnaJ-type J-protein, is present at over200-fold higher levels than either Apj1 or Xdj1, and under-scores the idea of functional specificity of Apj1 and Xdj1. Theresults presented here also demonstrate that Xdj1 and Apj1play their specialized roles as components of the Hsp70machinery. For both Xdj1 and Apj1, alteration of theJ-domain, which is required for Hsp70 activation, resulted inthe loss of both Apj1’s and Xdj1’s specialized activity.Moreover, the toxicity connected with modest overexpres-sion of Xdj1 is partially suppressed by simultaneous overex-pression of the nucleotide release factor Sse1, suggesting thatacceleration of the Hsp70-ATPase cycle subdues the toxiceffect.

How did these highly specialized J-proteins originate?Several lines of evidence suggest that the emergence ofXdj1 and Apj1 can be traced back to sequential duplicationsof an ancient YDJ1 gene, with both significantly predating thewhole genome duplication event that occurred in theSaccharomyces lineage approximately 100 Ma (Wolfe andShields 1997). Most likely, the first duplication that gave riseto Xdj1 took place in a common ancestor of Ascomycetes,whereas the second duplication event responsible for emer-gence of Apj1 was more recent and most likely took place in acommon ancestor of the Saccharomycetaceae and the CTGclade to which S. cerevisiae belongs. The presence of Xdj1orthologs in the Ascomycetes species and their absence inspecies outside the Ascomycota phylum, coupled with thepresence of Apj1 orthologs being restricted to species belong-ing to the Saccharomycetaceae and the CTG clade (Dujon2010), is consistent with this idea. In addition, the phyloge-netic reconstruction show that Xdj1, Apj1, and Ydj1 form astrongly supported monophyletic group and are more closely

Table 2. Amino Acid Sequence Divergence.

Group A Group B Residuesa Divergence Coefficients Type I Sitesd

?Ib ± SE ?II

c ± SE Invariant in A Invariant in B

Ydj1 Xdj1 317 0.48 ± 0.05 �0.46 ± 0.24 48 2

Ydj1post-Apj1 Apj1 325 0.39 ± 0.06 �0.37 ± 0.18 67 2

Ydj1post-Apj1 Ydj1pre-Apj1 363 0.26 ± 0.05 �0.11 ± 0.10 34 22

aResidues are the number of sites in the alignment.b�I is the coefficient of type I divergence.c�II is the coefficient of type II divergence.dType I sites are the number of sites in the alignment, which were invariant in the one group of sequences but variable in the other group (see Materials and Methods fordetails).

Table 3. Evolutionary Rates in Saccharomyces sensu stricto.

Gene dN dS dN/dS

YDJ1 0.006 ± 0.004 0.233 ± 0.148 0.027

XDJ1 0.039 ± 0.016 0.255 ± 0.104 0.155

APJ1 0.028 ± 0.014 0.349 ± 0.173 0.080

NOTE.—Values for dN and dS are shown as mean ± SD.

993

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

related to each other than to other members of DnaJ-familyfunctioning in endoplasmic reticulum (Scj1) or mitochon-drium (Mdj1). Moreover, in agreement with its more re-stricted phylogenetic distribution, and thus, likely youngerage, Apj1 forms a sister cluster to Ydj1, which strongly sup-ports the hypothesis that they are related by a gene duplica-tion event. We note that the topology of the protein tree doesnot directly support the origin of XDJ1 as a duplicate of YDJ1.However, we consider this evolutionary scenario very likely, asXDJ1 and YDJ1 coexist in all surveyed Ascomycetes speciesand that, based on protein phylogeny, XDJ1 and YDJ1 belongto the same clade.

The fact that XDJ1 and APJ1 were universally retained afterthey emerged points to the functional importance of theseJ-proteins and is consistent with our experimental analysesshowing that in S. cerevisiae both Xdj1 and Apj1 play special-ized roles in important cellular processes. Hints of whatdrives the specialized function of these two J-proteins canbe gleaned from the experimental data presented here.Evidence indicates that client protein binding is importantfor both. Xdj1’s peptide binding specificity is clearly differentfrom that of Ydj1. Moreover, in vivo tests indicated thatchanges within the peptide-binding cleft abolished the toxic-ity and defect in mitochondrial import caused by modestoverexpression. Although biochemical data available forApj1 are limited due to our inability to purify it in largeamounts, the results of our genetic analyses points to theconclusion that not only is client protein binding critical forfunction, but also the functional specificity of Apj1 is deter-mined by the domain containing the client-binding cleft.This idea is supported by the fact that neither the deletionof the GF-S region nor replacement of the Zn-binding regionby the Ydj1 homologous sequence affected Apj1’s ability tofunction. Although these data support the idea of specializedclient protein binding being behind the specificity, other ex-planations are possible. Indeed, localization of J-proteins toparticular sites of action is an established mode of specializa-tion (Kampinga and Craig 2010). That Xdj1 has been found inproteomic studies to be present in mitochondrial outer mem-branes supports this idea (Burri et al. 2006). Perhaps, bothclient-binding specificity and localization to the outer mito-chondrial membrane help to drive the functional specificityof Xdj1.

How do the specialized functions of Xdj1 and Apj1 relateto those of Ydj1? The results described in this report could beinterpreted according to the classical model of protein evo-lution purporting that after gene duplication, one copy of theduplicated gene preserves the original function (YDJ1), allow-ing the other (XDJ1 or APJ1) to be free to evolve new func-tions (Ohno 1970). At first glance, our data fits thisneo-functionalization model, as indeed, the rate of YDJ1 evo-lution was not affected by the presence of APJ1, and bothXDJ1 and APJ1 did evolve specialized functions. However, wefavor the alternative subfunctionalization scenario (Lynch andConery 2000) that Apj1 and Xdj1 took over functions alreadycarried out by multifunctional Ydj1. We hypothesize thatthese functions were fine-tuned, but not strictly new. Wenote that the broad biological functions in which Apj1 and

Xdj1 are involved, regulation of protein degradation andimport of proteins into mitochondria are ancient and onesin which Ydj1 is also known to function (Becker et al. 1996;Youker et al. 2004), albeit in different aspects of thesecomplex processes. An argument that may favor a neo-functionalization hypothesis for APJ1 and XDJ1 evolution istheir rapid sequence evolution. However, it is also well doc-umented that the rate of sequence evolution is reversely pro-portional to the protein expression level (Drummond et al.2005; Drummond and Wilke 2009), and thus the low expres-sion level in S. cerevisiae (and presumably other species) maywell explain the rapid rate. Moreover, the low and steady rateof YDJ1 evolution, which was not affected by the presence ofApj1, could well be due to a combination of its very highexpression level and significant functional constraints result-ing from its important biological roles.

In summary, based on the results of our functional andevolutionary analysis, we propose that the specialized func-tions of both Xdj1 and Apj1 evolved in two steps after geneduplication: 1) at an early stage, relaxation of selection due tolow expression of one of the duplicates allowed accumulationof changes that led to functional diversification and perhapsenhancement of a subset of specialized functions performedby the ancestral gene, and 2) at a later stage, purifying selec-tion maintained the newly specialized functions. Our resultshave implications for the functional expansion and diversifi-cation of chaperone networks, beyond the proteins function-ing in yeast cytosol. Interestingly, human cells also containthree closely related members of the DnaJ family in the cyto-sol/nuclear compartment (Vos et al. 2008). Similar to theiryeast counterparts, they are involved in many importantprocesses. Quite remarkably they also function in regulatedproteolysis and protein import to mitochondria (Terada et al.1997; Bhangoo et al. 2007; Fan and Young 2011). Thus, thefunctional diversification process based on gene duplicationand subsequent specialization described here might haveplayed an important role in formation of the taxon-specificHsp70–Hsp40 networks functioning in critical biologicalprocesses.

Materials and Methods

Ortholog Detection and Assignment

Using amino acid sequences of S. cerevisiae YDJ1, XDJ1, APJ1,SCJ1, and MDJ1 as queries, BLAST searches (Altschul et al.1990) were performed with fungal genomic data obtainedfrom NCBI, BROAD Institute, SGD, Genolevures, Podosporaanserina genome project, and JGI databases. A total of 43 spe-cies from the Ascomycetes, which were further subdividedinto three clades (Taphrinomycotina, Pezizomycotina, andSaccharomycotina) were analyzed (supplementary fig. S3,Supplementary Material online, for details).

To assess the presence or absence of particular DnaJ-likeorthologs, BLASTP and TBLASTN were used to identifyputative homologs within databases of annotated open read-ing frames (ORFs) and raw contig data for each species. Thegenomic locations of the best hit ORFs obtained with BLASTPwere verified to correspond with the location of the best

994

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

matches from the raw genomic data obtained with TBLASTN.If a high scoring hit could not be identified within the anno-tated ORFs, putative ORFs were identified directly from thegenomic region containing the best TBLASTN hit using ORFfinder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Hitswith a resulting E value of less than 10�10 were further in-spected for the presence or absence of specific sequencemotifs characteristic of the reference sequences from S. cere-visiae. Annotated results from each BLAST search were thenused as query sequences in a reciprocal BLASTP search againstthe S. cerevisiae protein database. We usedreciprocal-best-BLAST sequences as our operational defini-tion of orthologs.

Phylogenetic Tree Construction

Nucleotide and amino acid sequence data of YDJ1, XDJ1, APJ1,SCJ1, and MDJ1 orthologs were used to construct multiplesequence alignments for phylogenetic reconstruction. Toreduce method-specific bias, we used MAFFT v6.849b(Katoh et al. 2002) and Prank v121002 (Loytynoja andGoldman 2005) with default settings. The obtained align-ments were used to infer the phylogenetic trees using MLand Bayesian Inference (BI) methods. The ML analyses wereperformed using RaxML v.7.3.5 (Stamatakis 2006) with 100bootstrap replicates, under the LG (Le and Gascuel 2008)model of amino acid substitution, taking into account theempirical amino acid frequencies and using four categoriesderived from the gamma (g) distribution to estimate rateheterogeneity (LG + G). This model was estimated as thebest fitting substitution model by ProtTest v3.2 (Abascalet al. 2005). The BI analyses were performed using MrBayesv3.2 (Ronquist and Huelsenbeck 2003) under the WAGmodel (Whelan and Goldman 2001) of amino acid substitu-tion and assuming gamma distribution rate variation acrosssites (WAG + G model). This model was estimated byProtTest as the best fitting substitution model other thanthe LG model, which was not implemented in that versionMrBayes. The analyses were performed with two independentruns, each with four chains, running for 4,000,000 generationsand sampling every 100 generations. The first 25% of sampledtrees were discarded as burn-in before estimation of posteriorprobabilities for branch supports.

Sequence Analyses

Sequence identity and similarity scores were calculated as anarithmetic mean of all pairwise sequence identity/similaritycomparisons in a given multiple sequence alignment. Simi-larity scores were calculated as an arithmetic mean of allpairwise sequence similarity comparisons, using BLOSUM62for YDJ1 and BLOSUM35 for XDJ1 and APJ1. Two residueswere counted as similar if their substitution score in thecorresponding BLOSUM matrix was greater than 0.

Synonymous (dS) and nonsynonymous (dN) substitutionrates were determined for the YDJ1, XDJ1, and APJ1 orthologsusing the ML approach as implemented in the PAML packagev4.6 (Yang 2007) using the “codeml” program. A single dN/dSvalue was assumed for the entire tree obtained with ML

methods and mean dN and dS rates were obtained by aver-aging them across all branches of the tree.

Testing for lineage-specific changes in evolutionary ratesof YDJ1, XDJ1, and APJ1 in species belonging toSaccharomyces sensu stricto (S. cerevisiae, S. paradoxus,S. mikatae, S. kudriavzevii, and S. bayanus) (Dujon 2010)was performed as a series of likelihood ratio tests. Thelikelihood of a null hypothesis of a single dN/dS ratio onthe whole tree was compared against six alternativehypotheses. The first five hypotheses assumed a uniquedN/dS ratio on an external branch leading to a differentSaccharomyces sensu stricto species. The sixth hypothesisassumed that all branches of the Saccharomyces sensustricto tree have evolved under a different dN/dS ratio.

In all PAML analyses, the codon frequencies were averagedfrom nucleotide frequencies at the three codon positions(codon frequency model F3� 4). To avoid falling into sub-optimal likelihood peaks and ensure proper convergence,the analyses were carried out multiple times, using bothlow and high initial settings of the estimated parameters(dN/dS ratio and transition to transversion rate kappa, k).In each analysis, initial values of 0.1, 0.5, 1, and 5 were usedfor both parameters in all 16 different combinations.

Divergence Analyses

Coefficients of types I and II amino acid sequence divergence,as defined in (Gu 2001), were calculated using the DIVERGEsoftware (Gu and Vander Velden 2002). For type I changes,we used the likelihood-based method (Gu 2001), and allanalyses were bootstrapped on 100 replicates. Additionalsearches for sites exhibiting type I and type II divergencewas performed using a custom script written in BioPerl(Stajich et al. 2002). Multiple alignments of selected sequences(Ydj1:Xdj1, Ydj1postApj1:Ydj1preApj1, or Ydj1postApj1:Apj1) wereinspected for sites in strict agreement with the definition oftype I divergence, that is, invariant in one group of comparedsequences but variable in another group. To correct for rare,single time substitution events, the total count of type I siteswas reduced by the number of cases when a residue wasinvariant in all but a single sequence in the data set.

Codon Usage Bias

Codon usage bias was estimated using the effective numberof codons (ENC) (Wright 1990) and Codon Adaptation Index(CAI) (Sharp and Cowe 1991). Both statistics were calculatedusing the CodonW software (Peden 1999). ENC was calcu-lated for all surveyed species and CAI was calculated fororthologs from five closely related Saccharomyces sensu strictospecies (S. cerevisiae, S. paradoxus, S. mikatae, S. kudriavzevii,and S. bayanus) with the reference set of optimal codonsfrom S. cerevisiae.

Statistical Analyzes

Assessment of statistical significance of differences insequence identity/similarity values, as well as that of thedN and dS rates were performed in the R language forstatistical computation (R Development Core Team 2012).

995

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

Distributions of dN, dS, identity, and similarity values werefirst checked for nonnormality using the Shapiro–Wilk testand the level of statistical significance of difference betweenthem was assessed using the Kolmogorov–Smirnov test. The ttest for CAI values was performed as implemented in theLibreOffice Calc v3.5.4.2. All tests were done at P = 0.01significance.

Yeast Strains and In vivo Methods

All yeast strains used in this study were of the W303 geneticbackground. apj1�, xdj1�, ydj1�, and pam17� weredescribed previously (Sahi and Craig 2007; Schiller 2009).slx5� and slx8� strains were constructed by polymerasechain reaction (PCR) amplifying the KanMx gene cassettefrom the respective deletions strains of a knock-out library(Open Biosystems), using specific flanking primers and thentransforming the gene disruption cassette into W303. apj1�slx5� and xdj1� pam17� were constructed by crossing theparental yeast strains, tetrad dissection, and selecting forspores carrying the appropriate gene disruption markers.Cells were grown in glucose-based media.

Protein Purification

WT and variant Xdj1, Ydj1, and Apj1 were expressed as Smt3(SUMO)-His6 fusion proteins in the Rosetta E. coli strain(Roche). Induction was carried out at 18 �C for 18 h using0.5 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) in 1 l ofrich media having chloramphenicol (34mg/ml) andkanamycin (40mg/ml). Cells were chilled on ice beforepelleting. Cell pellets were dissolved in 30-ml ice-coldlysis buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid [HEPES], 0.5 M NaCl, glycerol [5%], 10 mMß-mercaptoethanol, 30 mM imidazole, pH 7.7) with proteaseinhibitor (complete Mini, ethylenediaminetetraacetic acid-free, Roche Diagnostics). Cells were lysed in a French Press,cleared by centrifugation at 14 k for 30 min. Ni–NTA His–Bind resin (2 ml) (Qiagen) equilibrated with the lysis bufferwas added to the clarified cell lysate and incubated with mildagitation for 2.5 h in the cold room. Ni resin was spun downand the supernatant was discarded. Resin was transferred to acolumn and sequentially washed with 25 ml of ice-cold lysisbuffer, 25 ml of lysis buffer with 1 M NaCl, and again with15 ml of lysis buffer. Proteins were eluted with 10 ml 0.5 Mimidazole in lysis buffer and dialyzed against 1 l of lysis bufferfor 3 h. Ulp1 protease, purified as described (Malakhov et al.2004), was added (at an approximate ratio to protein of1:100) directly into the dialysis bag (14 kDa cut off). Dialysiswas continued overnight. The protein was injected onto apre-equilibrated HiPrep 16/60 Sephacryl S-200 HR column(GE Healthcare) and eluted with 120 ml lysis buffer at aflow rate of 0.5 ml per min, 0.2 MPa. 1.4 ml fractions werecollected and analyzed on 10% sodium dodecyl sulphate(SDS)–polyacrylamide gel. Pooled protein fractions were con-centrated using Amicon Ultra centrifugal filters (Ultracel,3,000 MWCO, Millipore Corp.) and frozen in liquid nitrogenuntil further use.

For generating anti-Apj1 antibody, a C-terminal fragmentencoding the C-terminal 105 amino acids of Apj1 was PCRamplified with primers encoding a 50 BamHI and a 30 SacI siteand cloned into the pGEX-KG vector in frame as anN-terminal glutathionine-S-transferase fusion. For anti-Xdj1antibody, sequence encoding the C-terminal 210 residueswas PCR amplified with appropriate primers, digested withNdeI/BamHI and cloned into the same sites in proteinexpression vector pet3a. Apj1 and Xdj1 expression was in-duced with 0.5 mM IPTG for 3 h at 30 �C in C41 and Rosettacells, respectively. Protein was purified using glutathionineaffinity chromatography. Polyclonal antibodies were gener-ated in rabbits at Harlan laboratories (Madison, WI) usingpurified protein as antigen. For immunoblots, antibodieswere purified using HiTrap NHS-activated HP columns (GEHealthcare) and purified Apj1 and Xdj1 according to themanufacturers’ instructions.

Screening of the Phage Display Library

A phage display dodecapeptide library (Ph.D-12, New EnglandBiolabs) was used. 100mg of Xdj1 or Ydj1 was immobilized inindividual wells of polystyrene 96-well microtiter plates over-night in 0.1 N NaHCO3 pH 8.6 on a rocker shaker in the coldroom. The Hsp40-coated dish was then washed and blockedwith a solution that contained 5 mg/ml bovine serum albu-min (BSA) and 0.1 M NaHCO3 for 2 h at 4 �C. Subsequently,the plate was washed with Tris-buffered saline/Tween 20(TBS–T) (20 mM Tris–Cl, pH 8.0, 150 mM NaCl, 0.1%Tween 20) six times. 10ml of the Ph.D-12 library diluted to100ml in TBS–T was added in each Hsp40 coated well andincubated for 1 h at room temperature. The plate was thenwashed 10 times with TBS–T. Bound phage was eluted byaddition of 100ml of 0.2 M Glycine–HCl (pH 2.2), 1 mg/ml BSA for 20 min. The eluate was immediately neutralizedwith 15ml of 1 M Tris–HCl, pH 9.1. The eluted phage display-ing the peptides binding to Xdj1 or Ydj1 were amplified byinfection of a 20-ml culture of early log phase E. coli ER2537that was incubated for 4.5 h at 37 �C with vigorous shaking.The titer of the amplified phage was determined, and thesecond and third rounds of biopanning were performed.Later rounds were carried out as per manufacturer’s instruc-tions. After the third round of panning, the coliphage eluatewas plated. Coliphage present in isolated plaques was purifiedand the DNA sequenced to determine the sequence of18 peptides from each screening.

Plasmid Construction

The genomic clone of XDJ1 was constructed by PCR ampli-fying the coding sequence of Xdj1 along with 800 bp ofupstream and 400 bp of downstream sequence. The PCRfragment was digested with EcoRI/BamHI and cloned insame sites in pRS313. For making pRS314-Apj1, 400-bp up-stream and 400-bp downstream sequence was PCR amplifiedand cloned into XhoI/SacI sites in pRS314. CEN-ADH-Xdj1plasmid was constructed by subcloning the Xdj1 codingsequence from 2�-GPD-Xdj1 described previously (Sahiand Craig 2007) into the SpeI/XhoI sites in pRS414-ADH

996

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

vector. Internal deletions, pRS314-Apj1�79-161 (Apj1�G/F-S), pRS313-Xdj1�77-116 (Xdj1�GF), and all point muta-tions were generated by QuikChange PCR (Stratagene). ZnFRdeletion mutant pRS314-Apj1�206-262 (Apj1�Zn) was gen-erated similarly using primers that deleted the ZnFR and in-serted a GGSG linker. pRS314-Apj1-Zn Swap andpRS313-Xdj1-Zn Swap were made by designing primers thatwere complementary at their 50-ends to either Apj1 or Xdj1coding sequence just outside their respective ZnFR andamplifying the ZnFR (residues 143–206) of Ydj1. The resultingPCR fragment was used as a megaprimer for a QuikChangePCR to substitute the Zn finger in Apj1 and Xdj1 withthat of Ydj1. The complete coding sequences of Apj1, Xdj1,and Ydj1 were PCR amplified and digested with BamHI/XhoI and cloned into the same sites in pSmt3 (Gift fromW. Hendrickson, Columbia University) to generate pSmt3-Apj1, pSmt3-Xdj1, and pSmt3-Ydj1. pRS413-GPD-Sse1(Shaner et al. 2006) was a gift of K. Morano, Universityof Texas.

Other Methods

Extracts of total cellular proteins were prepared by treatingcells with 0.1 N NaOH and resuspension in SDS samplebuffer (62.5 mM Tris–HCl, pH 6.8, 5% glycerol, 2% SDS, 2%b-mercaptoethanol, and 0.01% bromophenol blue). Proteinwas detected by Enhanced Chemiluminescence kit(Amersham Pharmacia) according to manufacturer’s instruc-tions. SUMO-conjugates were resolved on a 4–15% gradientgel (BioRad) and probed with Anti-Smt3 polyclonal antibody(Gift from P. Hieter, University of British Columbia).Quantification of protein bands was performed with ImageJsoftware. Single point phage ELISA were performed in 96-wellpolystyrene microtiter plates essentially similar to the biopan-ning of the 12mer Ph.D library, except 2mg of protein wascoated and a single amplified phage clone was added in eachwell. Bound phage was detected with horseradish peroxidase-conjugated anti-M13 monoclonal antibody (GE Healthcare).Plates were visualized in a plate reader (Biotek) at 415 nm.Single-turnover ATPase assays of Ssa1 were carried out asdescribed previously (Lopez-Buesa et al. 1998). IsolatedHsp70-[�-32P]ATP complexes were incubated with 2-foldmolar excess of Apj1, Xdj1, or Ydj1 at 25 �C, and sampleswere removed for detection of ATP and ADP. The fractionsof ATP hydrolyzed to ADP over the time courses were quan-tified by PhosphorImager analysis (Molecular Dynamics) andthe data used to determine apparent rate constants for hy-drolysis of bound ATP.

Supplementary MaterialSupplementary figures S1–S7 are available at MolecularBiology and Evolution online (http://www.mbe.oxfordjournals.org/).

Acknowledgments

The authors thank Sanjith Reddy Yeruva for excellent tech-nical assistance, Chris T. Hittinger for thoughtful discussions,and Brenda Schilke for helpful comments on the manuscript.

They also thank Phil Heiter, Kevin Morano, and WayneHendrickson for antibody or expression plasmids. This workwas supported by the National Institutes of Health GM31107to E.A.C. and TEAM/2009-3/5 to J.M. and J.K.

ReferencesAbascal F, Zardoya R, Posada D. 2005. ProtTest: selection of best-fit

models of protein evolution. Bioinformatics 21:2104–2105.Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local

alignment search tool. J Mol Biol. 215:403–410.Becker J, Walter W, Yan W, Craig EA. 1996. Functional interaction of

cytosolic hsp70 and a DnaJ-related protein, Ydj1p, in protein trans-location in vivo. Mol Cell Biol. 16:4378–4386.

Bhangoo MK, Tzankov S, Fan AC, Dejgaard K, Thomas DY, Young JC.2007. Multiple 40-kDa heat-shock protein chaperones function inTom70-dependent mitochondrial import. Mol Biol Cell. 18:3414–3428.

Bukau B, Weissman J, Horwich A. 2006. Molecular chaperones andprotein quality control. Cell 125:443–451.

Burri L, Vascotto K, Gentle IE, Chan NC, Beilharz T, Stapleton DI,Ramage L, Lithgow T. 2006. Integral membrane proteins in the mi-tochondrial outer membrane of Saccharomyces cerevisiae. FEBS J.273:1507–1515.

Caplan AJ, Douglas MG. 1991. Characterization of YDJ1: a yeast homo-logue of the bacterial dnaJ protein. J Cell Biol. 114:609–621.

Caplan AJ, Tsai J, Casey PJ, Douglas MG. 1992. Farnesylation of YDJ1p isrequired for function at elevated growth temperatures inSaccharomyces cerevisiae. J Biol Chem. 267:18890–18895.

Cheetham ME, Caplan AJ. 1998. Structure, function and evolution ofDnaJ: conservation and adaptation of chaperone function. Cell StressChaperones 3:28–36.

Costanzo M, Baryshnikova A, Bellay J, et al. (53 co-authors). 2010. Thegenetic landscape of a cell. Science 327:425–431.

Craig EA, Huang P, Aron R, Andrew A. 2006. The diverse roles ofJ-proteins, the obligate Hsp70 co-chaperone. Rev Physiol BiochemPharmacol. 156:1–21.

Demoinet E, Jacquier A, Lutfalla G, Fromont-Racine M. 2007. The Hsp40chaperone Jjj1 is required for the nucleo-cytoplasmic recycling ofpreribosomal factors in Saccharomyces cerevisiae. RNA 13:1570–1581.

Dragovic Z, Broadley SA, Shomura Y, Bracher A, Hartl FU. 2006.Molecular chaperones of the Hsp110 family act as nucleotide ex-change factors of Hsp70s. EMBO J. 25:2519–2528.

Drummond DA, Bloom JD, Adami C, Wilke CO, Arnold FH. 2005. Whyhighly expressed proteins evolve slowly. Proc Natl Acad Sci U S A.102:14338–14343.

Drummond DA, Wilke CO. 2009. The evolutionary consequences oferroneous protein synthesis. Nat Rev Genet. 10:715–724.

Dujon B. 2010. Yeast evolutionary genomics. Nat Rev Genet. 11:512–524.Fan AC, Young JC. 2011. Function of cytosolic chaperones in

Tom70-mediated mitochondrial import. Protein Pept Lett. 18:122–131.

Fan CY, Ren HY, Lee P, Caplan AJ, Cyr DM. 2005. The type I Hsp40 zincfinger-like region is required for Hsp70 to capture non-native poly-peptides from Ydj1. J Biol Chem. 280:695–702.

Fitzpatrick DA, Logue ME, Stajich JE, Butler G. 2006. A fungal phylogenybased on 42 complete genomes derived from supertree and com-bined gene analysis. BMC Evol Biol. 6:99.

Glover JR, Lindquist S. 1998. Hsp104, Hsp70, and Hsp40: a novel chap-erone system that rescues previously aggregated proteins. Cell 94:73–82.

Gu X. 1999. Statistical methods for testing functional divergence aftergene duplication. Mol Biol Evol. 16:1664–1674.

Gu X. 2001. Maximum-likelihood approach for gene family evolutionunder functional divergence. Mol Biol Evol. 18:453–464.

Gu X, Vander Velden K. 2002. DIVERGE: phylogeny-based analysis forfunctional-structural divergence of a protein family. Bioinformatics18:500–501.

997

Sequential Duplications of a Member of DnaJ-Family . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

Hartl FU, Bracher A, Hayer-Hartl M. 2011. Molecular chaperones inprotein folding and proteostasis. Nature 475:324–332.

Kampinga HH, Craig EA. 2010. The HSP70 chaperone machinery: J pro-teins as drivers of functional specificity. Nat Rev Mol Cell Biol. 11:579–592.

Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel methodfor rapid multiple sequence alignment based on fast Fouriertransform. Nucleic Acids Res. 30:3059–3066.

Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T. 2009. TheSWISS-MODEL repository and associated resources. Nucleic AcidsRes. 37:D387–D392.

Le SQ, Gascuel O. 2008. An improved general amino acid replacementmatrix. Mol Biol Evol. 25:1307–1320.

Li J, Qian X, Sha B. 2003. The crystal structure of the yeast Hsp40 Ydj1complexed with its peptide substrate. Structure 11:1475–1483.

Li J, Sha B. 2005. Structure-based mutagenesis studies of the peptidesubstrate binding fragment of type I heat-shock protein 40. Biochem J.386:453–460.

Linke K, Wolfram T, Bussemer J, Jakob U. 2003. The roles of the two zincbinding sites in DnaJ. J Biol Chem. 278:44457–44466.

Lopez-Buesa P, Pfund C, Craig EA. 1998. The biochemical properties ofthe ATPase activity of a 70-kDa heat shock protein (Hsp70) aregoverned by the C-terminal domains. Proc Natl Acad Sci U S A.95:15253–15258.

Loytynoja A, Goldman N. 2005. An algorithm for progressive multiplealignment of sequences with insertions. Proc Natl Acad Sci U S A.102:10557–10562.

Lynch M, Conery JS. 2000. The evolutionary fate and consequences ofduplicate genes. Science 290:1151–1155.

Malakhov MP, Mattern MR, Malakhova OA, Drinker M, Weeks SD,Butt TR. 2004. SUMO fusions and SUMO-specific protease forefficient expression and purification of proteins. J Struct FunctGenomics. 5:75–86.

Mayer MP. 2010. Gymnastics of molecular chaperones. Mol Cell. 39:321–331.

Meyer AE, Hung NJ, Yang P, Johnson AW, Craig EA. 2007. The special-ized cytosolic J-protein, Jjj1, functions in 60S ribosomal subunitbiogenesis. Proc Natl Acad Sci U S A. 104:1558–1563.

Mullen JR, Brill SJ. 2008. Activation of the Slx5-Slx8 ubiquitin ligase bypoly-small ubiquitin-like modifier conjugates. J Biol Chem. 283:19912–19921.

Ohno S. 1970. Evolution by gene duplication. New York: Springer.Pais JE, Schilke B, Craig EA. 2011. Reevaluation of the role of the

Pam18:Pam16 interaction in translocation of proteins by the mito-chondrial Hsp70-based import motor. Mol Biol Cell. 22:4740–4749.

Pan X, Ye P, Yuan DS, Wang X, Bader JS, Boeke JD. 2006. A DNA integritynetwork in the yeast Saccharomyces cerevisiae. Cell 124:1069–1081.

Peden JF. 1999. Analysis of codon usage [PhD thesis]. [Nottingham(UK)]: University of Nottingham.

R Development Core Team. R: a language and environment for statis-tical computing [Internet]. Vienna, Austria: R Foundation forStatistical Computing; 2012. R version 2.15.1 (2012-06-22). ISBN3-900051-07-0. Available from: http://www.R-project.org.

Raviol H, Sadlish H, Rodriguez F, Mayer MP, Bukau B. 2006. Chaperonenetwork in the yeast cytosol: Hsp110 is revealed as an Hsp70nucleotide exchange factor. EMBO J. 25:2510–2518.

Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogeneticinference under mixed models. Bioinformatics 19:1572–1574.

Sahi C, Craig EA. 2007. Network of general and specialty J protein chap-erones of the yeast cytosol. Proc Natl Acad Sci U S A. 104:7163–7168.

Sahi C, Lee T, Inada M, Pleiss JA, Craig EA. 2010. Cwc23, an essential Jprotein critical for pre-mRNA splicing with a dispensable J domain.Mol Cell Biol. 30:33–42.

Schiller D. 2009. Pam17 and Tim44 act sequentially in protein importinto the mitochondrial matrix. Int J Biochem Cell Biol. 41:2343–2349.

Shaner L, Sousa R, Morano KA. 2006. Characterization of Hsp70 bindingand nucleotide exchange by the yeast Hsp110 chaperone Sse1.Biochemistry 45:15075–15084.

Sharp PM, Cowe E. 1991. Synonymous codon usage in Saccharomycescerevisiae. Yeast 7:657–678.

Stajich JE, Block D, Boulez K, et al. (21 co-authors). 2002. The Bioperltoolkit: Perl modules for the life sciences. Genome Res. 12:1611–1618.

Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylo-genetic analyses with thousands of taxa and mixed models.Bioinformatics 22:2688–2690.

Summers DW, Douglas PM, Ren HY, Cyr DM. 2009. The type I Hsp40Ydj1 utilizes a farnesyl moiety and zinc finger-like region to suppressprion toxicity. J Biol Chem. 284:3628–3639.

Terada K, Kanazawa M, Bukau B, Mori M. 1997. The human DnaJhomologue dj2 facilitates mitochondrial protein import and lucifer-ase refolding. J Cell Biol. 139:1089–1095.

van der Laan M, Chacinska A, Lind M, Perschil I, Sickmann A, Meyer HE,Guiard B, Meisinger C, Pfanner N, Rehling P. 2005. Pam17 is requiredfor architecture and translocation activity of the mitochondrialprotein import motor. Mol Cell Biol. 25:7449–7458.

Vos MJ, Hageman J, Carra S, Kampinga HH. 2008. Structural andfunctional diversities between members of the human HSPB,HSPH, HSPA, and DNAJ chaperone families. Biochemistry 47:7001–7011.

Walsh P, Bursac D, Law YC, Cyr D, Lithgow T. 2004. The J-protein family:modulating protein assembly, disassembly and translocation. EMBORep. 5:567–571.

Whelan S, Goldman N. 2001. A general empirical model of proteinevolution derived from multiple protein families using amaximum-likelihood approach. Mol Biol Evol. 18:691–699.

Wolfe KH, Shields DC. 1997. Molecular evidence for an ancient dupli-cation of the entire yeast genome. Nature 387:708–713.

Wright F. 1990. The “effective number of codons” used in a gene. Gene87:23–29.

Yang Z. 2007. PAML 4: phylogenetic analysis by maximum likelihood.Mol Biol Evol. 24:1586–1591.

Youker RT, Walsh P, Beilharz T, Lithgow T, Brodsky JL. 2004. Distinctroles for the Hsp40 and Hsp90 molecular chaperones during cysticfibrosis transmembrane conductance regulator degradation in yeast.Mol Biol Cell. 15:4787–4797.

Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, Krogan N,Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA. 2005.Navigating the chaperone network: an integrative map of physicaland genetic interactions mediated by the hsp90 chaperone. Cell 120:715–727.

998

Sahi et al. . doi:10.1093/molbev/mst008 MBE at U

niversity of Wisconsin M

adison - General L

ibrary System on M

ay 20, 2013http://m

be.oxfordjournals.org/D

ownloaded from

Supplementary  Figure    S1.  Alignment  of  S.  cerevisiae  Ydj1,  Xdj1  and  Apj1  amino  acid  sequences.  The  Ydj1,  Xdj1  and  Apj1  alignment  is  based  on  the  mul9ple  sequence  alignment  of  all  107  sequences  analyzed  in  this  study:  43  for  Ydj1,  43  for  Xdj1  and  21  for  Apj1.  The  domain  boundaries,  based  on  Ydj1,  are  shown  above  the  alignment.  The  C-­‐  terminal  prenyla9on  mo9fs  (CXXQ)  in  Ydj1  and  Xdj1  are  marked  by  black  boxes.  (*):  residues  invariant  across  all  analyzed  sequences.  Red  background:  most  conserved  –  residues  are  iden9cal  or  similar  (based  on  the  BLOSUM62  matrix)  for  all  sequences  belonging  to  a  given  orthologous  group  (Ydj1,  Xdj1  or  Apj1).  Yellow  background:  conserved  residues  that  do  not  reach  the  bar  for  red  coloring,  but  are  iden9cal  or  similar  (based  on  the  BLOSUM  62  matrix)  in  the  14  species  of  Saccharomyceteacae  analyzed.  Residues  were  not  colored  if  more  than  2  non-­‐matching  (neither  iden9cal  nor  similar)  residues  were  found  in  any  given  comparison.  

Supplementary Figure S2. Ydj1 is approximately 100 times more abundant that Apj1 and Xdj1:. ( A, B,C) Indicated amounts (OD600)of total protein isolated from wild-type, or the indicated mutant, yeast cells and purified Ydj1, Xdj1 or Apj1 (ng) were resolved by electrophoresis, electro-blotted and probed with antibody specific for one of the J-proteins, as indicated. Blots were scanned and densitometric quantification was performed using ImageJ software. (A) only wt lysates. (B) lysates from xdj1Δ, wild-type (WT) and xdj1Δ cells expressing pRS414-ADH-Xdj1 (ADH-Xdj1). All samples were run on a single gel and blotted and processed together. Dashed line indicates where irrelevant lanes were spliced out. C) wild-type (WT) or apj1∆ yeast. Levels of protein in wt cells, estimated as described in Ghaemmaghami et al, 2003, were approximately 90,000, 500 and 100 molecules/cell of Ydj1, Xdj1 and Apj1, respectively. The estimates reported by Ghaemmaghami et al, 2003 using tagged protein in a high throughput analysis were 119,000, 1210 and 125 molecules/cell.

0.00

5

0.00

2

Yeast Lysate OD600 Purified Ydj1 (ng)

4 0.8

0.16

0.03

2

A

Ydj1

Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, Dephoure N, O'Shea EK, Weissman JS (2003) Global analysis of protein expression in yeast. Nature 425: 737-741.

C

0.05

0.1

0.2

0.5

1 2

Purified Apj1 (ng) WT apj1∆

Yeast lysate OD600

Apj1

0.05

0.1

0.2

1 2 4

Xdj1

Purified Xdj1 (ng) B

Xdj

1∆

WT

AD

H-

Xdj

1

Yeast lysate OD600 0.1

Species 4-letter code ClassificationSchizosaccharomyces japonicus Sjap TaphrinomycotinaSchizosaccharomyces pombe Spom TaphrinomycotinaSchizosaccharomyces octosporus Soct TaphrinomycotinaSchizosaccharomyces cryophilus Scry TaphrinomycotinaAjellomyces capsulatus Acap PezizomycotinaUncinocarpus reesii Uree PezizomycotinaCoccidioides immitis Cimm PezizomycotinaAspergillus nidulans Anid PezizomycotinaAspergillus fumigatus Afum PezizomycotinaAspergillus oryzae Aory PezizomycotinaAspergillus terreus Ater PezizomycotinaPhaeospheria nodorum Pnod PezizomycotinaMagnaporthe grisea Mgri PezizomycotinaNeurospora crassa Ncra PezizomycotinaPodospora anserina Pans PezizomycotinaChaetomium globosum Cglo PezizomycotinaTrichoderma reesei Tree PezizomycotinaGibberella moniliformis Gmon PezizomycotinaGibberella zeae Gzea PezizomycotinaBotryotinia fuckeliana Bfuc PezizomycotinaSclerotinia sclerotiorum Sscl PezizomycotinaYarrowia lipolytica Ylip YarrowiaCandida lusitaniae Clus CTG groupPichia guilliermondii Pgui CTG groupDebaryomyces hansenii Dhan CTG groupCandida parapsilosis Cpar CTG groupCandida tropicalis Ctro CTG groupCandida dubliniensis Cdub CTG groupCandida albicans Calb CTG groupLachancea waltii Lwal pre-WGDLachancea kluyveri Lklu pre-WGDLachancea thermotolerans Lthe pre-WGDEremothecium gossypii Egos pre-WGDKluyveromyces lactis Klac pre-WGDZygosaccharomyces rouxii Zrou pre-WGDVandewaltozyma polyspora Vpol post-WGDSaccharomyces castelli Scas post-WGDCandida glabrata Cgla post-WGDSaccharomyces bayanus Sbay post-WGDSaccharomyces kudriavzevii Skud post-WGDSaccharomyces mikatae Smik post-WGDSaccharomyces cerevisiae Scer post-WGDSaccharomyces paradoxus Spar post-WGD

Supplementary Figure S3. List of Ascomycetes species surveyed in this work.

Supplementary Figure S5. Dodecapeptides presented on phage display isolates recovered in biopanning experiments with Xdj1 or Ydj1. The letter prefix in each ID number (left column) indicates the protein target used (X for Xdj1, Y for Ydj1). The deduced amino acid sequence of the peptide displayed at the N-terminus of M13 coat protein III is displayed in the center column. Frequency refers to the number of independent isolates obtained for each specific sequence.

 Xdj1          Hsp82  

WT                WT  WT          G170D  xdj1Δ            WT  xdj1Δ      G170D  

23°C   30°C   34°C  

Supplementary Figure S6. No obvious synthetic genetic interaction of xdj1∆ with Hsp90 mutant. 5µl of ten fold serial dilution of hsc82Δ hsp82Δ or xdj1Δ hsc82Δ hsp82Δ cells expressing either pRS314-GPD-Hsp82 (WT) or the ts allele of Hsp90, pRS314-GPD-Hsp82G170D (G170D) were spotted on Trp omission plates and incubated at indicated temperatures for 3 days. Similar results (data not shown) were found using the strain background in which the original genomic wide screen was performed (Zhao R et al 2005).

Zhao R, Davey M, Hsu YC, Kaplanek P, Tong A, Parsons AB, Krogan N, Cagney G, Mai D, Greenblatt J, Boone C, Emili A, Houry WA (2005) Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone. Cell 120: 715-727

Supplementary Figure S7. Fully resolved maximum-likelihood tree of the amino acid sequences of Ydj1, Xdj1, Apj1, Scj1 and Mdj1 used in the main text as Figure 6. All bootstrap values are shown above their respective branches. Species names and their codes are listed in Fig. S1.