Initial characterization of the nascent polypeptide-associated complex in yeast

Yeast 15, 397–407 (1999)

Initial Characterization of the NascentPolypeptide-Associated Complex in Yeast

BARBARA REIMANN1, JOHN BRADSHER2, JACQUELINE FRANKE1, ENNO HARTMANN3,MARTIN WIEDMANN2, SIEGFRIED PREHN1 AND BRIGITTE WIEDMANN1*1Humboldt-University, Charite, Department of Biochem., Hessische-Str. 3–4, 10115 Berlin, Germany2Memorial Sloan-Kettering Cancer Center, Cellular Biochemistry and Biophysics Program, 1275 York Avenue,New York, NY 10021, U.S.A.3MDC, R.-Roessle-Str. 10, 13125 Berlin-Buch, Germany

The three subunits of the nascent polypeptide-associated complex (á, â1, â3) in Saccharomyces cerevisiae are encodedby three genes (EGD2, EGD1, BTT1). We found the complex bound to ribosomes via the â-subunits in asalt-sensitive manner, in close proximity to nascent polypeptides. Estimation of the molecular weight of the complexof wild-type cells and cells lacking one or two subunits revealed that the composition of the complex is variable andthat as yet unknown proteins might be included. Regardless of the variability, a certain balance of the subunits hasto be maintained: the deletion of one subunit causes downregulation of the remaining subunits at physiologicalgrowth temperature. Cells lacking both â-subunits are unable to grow at 37)C, most likely due to a toxic effect of theá-subunit. Based on in vitro experiments, it has been proposed that the function of mammalian nascent-polypeptideassociated complexes (NAC) is to prevent inappropriate targeting of non-secretory nascent polypeptides. In vivo,however, the lack of NAC does not cause secretion of signal-less invertase in yeast. This result and the lack of adrastic phenotype of cells missing one, two or three subunits at optimal conditions (28)C, YPD-medium) suggesteither the existence of a substitute for NAC or that cells tolerate or ‘repair’ the damage caused by the absence ofNAC. Copyright ? 1999 John Wiley & Sons, Ltd.

— NAC; Saccharomyces cerevisiae; protein synthesis; nascent-polypeptide-associated complex

*Correspondence to: B. Wiedmann, Department of Bio-chemistry, Humboldt-University, Charite, Hessische-Str. 3–4,10115 Berlin, Germany. Tel.: 030 2093 7213; fax: 030 2093

INTRODUCTION

The nascent polypeptide-associated complex wasoriginally isolated from bovine brain cytosol as anabundant protein complex of á- and â-subunits.NAC is one of the first cytosolic proteins describedthat interacts with the nascent polypeptide pro-truding from the ribosome (Wiedmann et al.,1994). NAC protects the nascent peptide frompremature interaction with other cytosolic proteinsuntil it is long enough to be bound by the correctpartner and guided to its final destination site(Wang et al., 1995; Wickner, 1995; Neupert andLill, 1994; Lauring et al., 1995a, b). Whenribosome-nascent polypeptide-complexes (RNCs)are stripped of all cytosolic proteins, includingNAC, they can bind to the ER-membrane and

7217; e-mail: [email protected]

CCC 0749–503X/99/050397–11 $17.50Copyright ? 1999 John Wiley & Sons, Ltd.

translocation occurs even of peptides lackinga signal sequence, albeit with low efficiency(Wiedmann et al., 1994; Lauring et al., 1995a, b).Ribosomes themselves have an intrinsic affinity formicrosomes. The activity of several cytosolic pro-teins (e.g. NAC, SRP, HSPs) guarantees thatonly ribosomes translating proteins with signalsequence bind to the membrane (Lauring et al.,1995a, b; Wickner, 1995; Millmann et al., 1997;Powers and Walter, 1996). Furthermore, recentinvestigations (Moller et al., 1998) furthersupport the original hypothesis that NAC masksa membrane attachment site (M-site) on theribosome.

Since these results were all derived from in vitroexperiments with factors of heterogeneous origin(bovine NAC, canine SRP, ribosomes fromwheat germ or reticulocytes, etc.) we decided toinvestigate NAC’s function and structure in a
homogeneous system, using yeast.
Received 2 July 1998Accepted 9 November 1998

398 . .

While the known number of NAC genes andproteins in humans is still growing, there are onlythree genes (EGD2/á-subunit, EGD1/â1-subunit,BTT1/â3-subunit) on different chromosomesknown in yeast. We have used single, doubleand triple knockout strains as well as strainsoverexpressing a subunit in our investigation.

MATERIALS AND METHODS

Strains and plasmidsExperiments were started with knockout

mutants of Saccharomyces cerevisiae strain W303(MATa ade2 his3-11,15 leu2-3,113 trp1-1 ura3-1)that were constructed in the laboratory of Dr. H.Ronne by insertion of an amino acid marker geneinto unique restriction sites of the genes (Hu andRonne, 1994). We replaced those mutants withknockout mutants constructed in our laboratoryaccording to Schneider et al. (1996) in order tomaintain selection markers and to destroy thepromoter region. We removed about 1000 bp ofthe promoter and coding region. The two differentknockout mutants of the same gene(s) had thesame phenotype.

The NAC-genes were isolated by PCR froma yeast cDNA library, â3-NAC was cloned intovector p2UG (Guthrie and Fink, 1991), á- andâ1-NAC into the constitutive vector pSEY (Emret al., 1986). The cytoplasmic and secreted forms ofinvertase were expressed from plasmid pRS 414(Sikorski et al., 1989) containing a GAL10 pro-moter into which they were recloned from plasmidspBR420 and 576 (Carlson and Botstein, 1982).

Yeast was transformed by the LiOAc–method(Kaiser et al., 1994).

Secretion of invertaseThis was tested by the ability of the yeast cells

to grow on YPsucrose. The cells of O/N cultureson YPD were counted, adjusted to equivalentdensities, and 5 ìl of consecutive dilutions weredropped onto YPsucrose plates which containedthe pH indicator bromothymol blue. Only cellsthat secrete invertase are able to form colonies onthis medium and cause a colour change from greento yellow due to the production of protons (Kaiseret al., 1994).

Cell fractionation, sizing column, and preparationof in vitro translation lysates

Cells were grown to middle log-phase on YPD
(or SD when plasmids had to be maintained). They
Copyright ? 1999 John Wiley & Sons, Ltd.

were spheroplasted with Zymolyase 100T, hom-ogenized and centrifuged as described (Wiedmannet al., 1988) with the exception that the separationof cytosol and microsomes occurred at 40 000#g.To prepare a translation lysate the supernatantwas applied to a Sephadex G25 column and frac-tions of the highest OD260 were pooled and frozenas droplets in liquid nitrogen.

Ribosomes were sedimented in a Beckman TL100 rotor at 100 000 rpm for 30 min. NAC wasisolated by washing the sedimented ribosomes in500 m KOAc with the exception of the â1-knockout strains, where the remaining subunitsstay cytosolic and can be applied directly to asizing column. The stripped ribosomes were sedi-mented again and 200 ìl of the supernatant wasapplied either directly or after dialysis against alow-salt buffer to a Superdex-200 column.

Partial purification of the complexRibosomes of 27 l yeast culture were incubated

with 500 m KOAc to release NAC which wasfound then in the supernatant of a 400 000#gcentrifugation (1 h, TL 100.4-rotor, Beckman).The supernatant was applied first to a heparin- andthen to a Mono-Q-Sepharose column.

Co-immunoprecipitationCells were grown to middle log-phase, sphero-

plasted and homogenized in IP-buffer withoutdetergents (100 m potassium phosphate, 150 mNaCl, 1 m PMSF). Cell debris and nuclei wereremoved by centrifugation at 3000#g for 10 minand, subsequently, mitochondria by centrifugationof the first supernatant at 10 000#g for 15 min.One ml of the resulting supernatant (about 7 mgprotein) was incubated for 1 h at room tempera-ture and O/N at 4)C with 3 ìl antibodies. Antigen–antibody complexes were collected with 50 ìl 50%protein A-sepharose in IP-buffer, washed exten-sively with IP-buffer, and solubilized by boiling in50 ìl SDS sample buffer.

Electrophoresis and Western blottingProteins were separated by 12% PAGE with

0·7% bisacrylamide in order to separate theâ-subunits. Western blot detection occurred withthe ECL system according to the instructions ofthe manufacturer (Amersham). Gels for fluorogra-phy were fixed in 10% acetic acid/40% methanol

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for 10 min, incubated in 1 sodium salicylate,dried and exposed to KODAK XOMAT films at"80)C.

PhotocrosslinkingPhotocrosslinking of proteins to nascent

polypeptides was performed according to Gorlichet al. (1991) in 100 ìl translation assays. Ribo-somes were pelleted, resuspended in 100 ìlIP-buffer w/o detergents and treated with RNaseA. SDS was added to 1%, and the samples wereboiled twice to inactivate the RNase. After 10-folddilution with IP-buffer w/o detergents, one-quarterof the sample was used for immunoprecipitationwith each antibody.

Growth curvesStrains were grown O/N at 30)C on YPD,

diluted to about 0·03 OD600 into YPD and grownat the indicated temperature. Divisions were calcu-lated from OD600. To test the influence of heatshock on growth on synthetic medium, cells wereprecultured on SD medium with necessary aminoacids O/N at 30)C or 37)C and then diluted toabout 0·03 OD600 and grown at 30)C and 37)C. Alldiagrams represent the average of at least threeexperiments.


RESULTS

Figure 1. Subunits of NAC co-purify. NAC was enriched by sedimentation of theribosomes, then extracted in a 500 mM KOAc buffer. The high-salt supernatant wasapplied to a Hi Trap heparin column and further purified on a Mono-Q-Sepharose column.Fractions from the Mono-Q-Sepharose column were analysed in parallel by Coomassiestain and by Western blot with anti-á NAC. The indicated proteins (arrows) were identifiedas á- and â-NAC by peptide sequencing.

Composition and localization of NACHere we show for the first time that NAC, an

abundant cytosolic protein complex in highereucaryotic cells, is also present in yeast cells. Usinga variety of genetic and biochemical approaches,we demonstrate that yeast NAC is also aribosome-associated protein. During the partialpurification of the á-subunit by heparin- andMono-Q-Sepharose column chromatography, wefound that the â1-subunit eluted in the same frac-tions as the á-subunit (Figure 1, arrows). Theidentity of the eluting proteins was established bymicrosequencing. In wild-type yeast, more than95% of the NAC is ribosome-bound, as shown byco-sedimentation analysis (Figure 2, WT, lane R).In a strain carrying a â1-knockout mutation, theá-subunit remains cytosolic, indicating that â1mediates the binding of the NAC to ribosomes(Figure 2, Äâ1, Äâ1â3, lane C). The cellular con-centration of the â3-subunit in our strain is twoorders of magnitude lower than that of the othertwo subunits (our results, paper in preparation).The â3-protein is only detectable by Western blot-ting when the gels are highly overloaded or whenthe lysate is prepared from a strain overexpressingâ3 from an inducible plasmid. Also, when onesubunit is deleted the amount of the other is

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Figure 2. The â1-subunit can mediate ribosome association of NAC. Wild-type yeast(WT), different knockout strains, and â3-overexpressing strains (in order to be able to detectthe protein in Western blots) were grown to log-phase, spheroplasted with zymolyase andlysed in hypo-osmotic buffer. Cytosol and ribosomes were separated by a 100 000#gcentrifugation for 30 min after removal of cell debris, nuclei and mitochondria. Lysate (L)and cytosol (C) of the indicated strains were TCA-precipitated and resuspended in the samevolume of sample buffer as ribosomes (R) and analysed by Western blot. The amount ofprotein per lane (50–150 ìg) and the exposure time (20 s–5 min) were adjusted as necessaryto detect the subunits. We used affinity purified antibodies against the â1- and â3-subunitsand a cross-reacting serum against the human á-subunit.

Figure 3. Subunits of the NAC co-immunoprecipitate. Wild-type yeast (WT) and â1-knockout strains containing an inducible â3 gene were grown to log-phase. â3-NACexpression was induced with deoxycorticosterone and after 3·0 h the cells were harvested andtreated as described in Methods. The immunoblot was probed with affinity purifiedantibodies against the â1- and â3-subunits and a cross-reacting serum against the humaná-subunit; ‘pre’ stands for pre-immune serum of the â1 antibody.

diminished. This made it necessary to vary theprotein amounts from strain to strain in order todetect the subunits by Western blotting (see alsoFigure 3: comparison of the á-NAC concentrationin WT- and â1-knockout strains).The â3-subunit isable to mediate binding of the á-subunit to theribosomes, which is obvious in a â1-knockoutback-ground with â3 overexpressed (Figure 2,Äâ1+â3, lane R) and from results of co-immunoprecipitation experiments (Figure 3).Using co-immunoprecipitation we were able todemonstrate that the á- and â -subunits form a
1

complex (Figure 3, lanes 2, 3, 6 and 7) and that theá- but not the â1-subunit can be co-precipitatedwith the â3-protein when the latter protein isoverexpressed (Figure 3, lanes 4 and 8). Resultsshown in Figures 2 and 3 indicate that bothâ-proteins are separately in contact with theá-subunit and can each separately mediateribosome association of the complex.

The three subunits always co-migrate on a sizingcolumn (Figure 4). Again, â3 is only detectable inWT or in mutant strains when overexpressed. Theamount of the á- and â -subunit in strains missing
1
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the opposite subunit is less than in WT, indicatingcoordinated regulation of protein expression levelsof these subunits. The á-subunit appears often as adoublet in Western blots; the smaller band is likelyto be a degradation product of the protein. Thesize of the NAC-containing complexes ranges from45 kDa (=ovalbumin, fraction 31) to over 68 kDa(=albumin, fraction 27) in wild-type and Äâ-strains. It is reduced to about 45–50 kDa when theá-subunit is deleted and increases dramaticallyin the double â-knockout. We did not find anyimmunoreactive material above fraction #32 (pro-teins smaller than ca. 30 kDa), indicating that themajority of the NAC-subunits are in a complex.

In analogy to previous experiments performedby us in reticulocyte lysate, we tested whether theyeast NAC-subunits are in contact with nascentpolypeptides (Wiedmann et al., 1994; Lauringet al., 1995a, b). For this we used a photocrosslink-ing approach where the crosslinker is positioned inthe nascent chain itself (Gorlich et al., 1991). TheN-terminal 86 amino acids of preprolactin (86aapPL) and 72 amino acids of chloramphenicolacetyltransferase (72aa CAT) were used as nascentpeptides which have or lack a signal sequence,respectively. Both peptides were found to becrosslinked to á- and â1-NAC after translation inwild-type yeast lysate (Figure 5, lanes 3 and 5).Immunoprecipitation with antibodies against â3-NAC did not show any radioactive labelledcrosslink product (data not shown), most likelydue to the low abundance of â3-NAC itself (seeFigure 2).

Figure 4. Size of NAC in strains which are deleted of differentNAC-subunits. NAC-containing cytosol of the indicatedstrains was prepared (as described in the methods) and samplesof 400 ìg–1 mg protein were applied to a Superdex 200 column.Fractions of 0·5 ml were TCA precipitated and analysed byPAGE and Western blotting. The column was calibrated withalbumin (eluted in fraction 27), ovalbumin (fraction 31) andchymotrypsinogen (fraction 36).

Effect of NAC deletion or overexpressionGrowth of all possible combinations of knock-

out mutants was tested on YP with glucose, galac-tose or sucrose as carbon source at 23)C, 30)C,37)C and 43)C (for growth on glucose, see Figure6A and data not shown). Even deletion of all threesubunits did not influence growth at 23)C and30)C. However, the mutant strains divided lessfrequently under heat shock conditions (43)C)after 5 h of similar growth to the wild-type strain.The effect of the heat shock became more evidentafter prolonged cultivation, especially on a syn-thetic complete medium: the double â-knockoutstrain stopped dividing (Figure 6B). It was not ableto grow at all on SD agar at 37)C (Figure 6D).Cells which were grown overnight at 37)C startedto divide again only at about 24 h after transfer to28)C. The triple knockout strain was not as

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402 . .

strongly impaired. We concluded that excess of theá-subunit caused cell death when not in complexwith one of the â-subunits. Such a situation mightonly occur under the stress condition of heatshock. To test this we overexpressed á-NAC inwild-type cells and found that this strain alsoshows impaired growth at 37)C (Figure 6C).

As NAC has been implicated in targeting ofpolypeptides to their correct location in the cell, wetested the possibility that in NAC knockout strainspreproteins accumulate or that heat shock proteinsare induced to cope with the possible existence ofmislocalized proteins. Wild-type and mutantstrains were grown at 30)C and 37)C/43)C andsamples taken every 30 min for 4 h. The cells wereboiled in an equal volume of two-fold Laemmlisample buffer and immunoblots were probed withantibodies against the NAC-subunits, YDJ1, SSA,


SSB, KAR2 and HSP60. There were no differencesdetectable between the strains and no precursoraccumulation occurred (data not shown). In asecond set of experiments, we fractionated the celllysates after zymolyase-lysis and searched forHSP60 precursor accumulation in the cytosol andoccurrence of unfarnesylated YDJ1 in NAC-mutants compared to wild-type cells. Again, wecould not detect any differences (Figure 7).

Finally we tested whether the occurrence ofmistargeting in a mammalian in vitro system isdetectable in yeast in vivo. We knocked out theSUC2 gene in different NAC knockout mutantsand constructed two plasmids encoding wild-type(secreted) invertase and signalless (cytosolic)invertase. Expression of both proteins was testedby Western blotting (data not shown). Cells ofall tested strains expressing the signal-bearinginvertase were able to grow on sucrose or raffinoseas the only carbon source as well as the corre-sponding SUC2+ strain. The cytosolic form ofinvertase (without a signal sequence) was notsecreted, or secreted in an insufficient amount topromote growth on sucrose (Figure 8) or raffinose,independent of the genetic background.

Figure 5. NAC is in close proximity to nascent polypeptides.86 aa pPL and 72 aa CAT were translated in WT-lysate andcross-linked. The ribosomes were washed with 500 m KOAcand sedimented. The pellet was resuspended in 50 m Tris,pH 8·8, and treated with 20 ìg RNase A/100 ìl for 10 min. SDSwas added to a final concentration of 1%, the samples wereboiled twice, diluted 10-fold with IP-buffer lacking detergentsand then processed for immunoprecipitation.

DISCUSSION

Three NAC-genes with homology to mammaliangenes have been identified in yeast: EGD2, thehomologue of á-NAC, and EGD1 and BTT1,homologues of â-NAC. As in mammals, the á- andâ-subunits form also in yeast a salt-resistant com-plex (see co-immunoprecipitation, Figure 3 andpartial purification, Figure 1). Analysis of thelocation of NAC-proteins in the different knock-out strains revealed that the salt-sensitive ribosomeassociation of the complex is mediated by theâ1-subunit (Figure 2). Using a well-establishedphotocrosslinking approach we demonstrated theproximity of yeast NAC to nascent polypeptidesanalogous to mammalian NAC (Figure 5). There-fore, we assume the main function of this complexin yeast to be linked to the ribosome as it is inmammals.

The â3-subunit behaves similarly to the â1-subunit: it is found at the same location (Figure 2),it interacts with the á-subunit (Figure 3), and it issufficient to mediate the binding of the complex tothe ribosome (Figure 2). As we did not find anyconditions where the â3-subunit is upregulatedin wild-type cells, its function remains open.

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Figure 6. Growth of WT and knockout strains on YPD at different temperatures. (A) Growth onrich medium (YPD) at different temperatures. (B) Growth on a synthetic complete medium at 37)Cafter 12 h of preculture at 37)C. (C) Strains were grown on synthetic complete medium at 37)C for12 h, diluted, and grown an additional 8 h at 37)C. The growth of the WT-strain at 30)C was setat 100%. (D) Growth on synthetic complete-agar plates at 30)C and 37)C.

Interestingly, only the double â-knockout, not theâ1-knockout alone, exhibits a phenotype whengrown for a longer period at 37)C (Figure 6). Thisis in conjunction with the lethal phenotype of theâ-knockout mutants in Drosophila and mice (Dengand Behringer, 1995; Markesich and Beckingham,1997). In these organisms, one â-gene codes forseveral â-proteins, which are generated by differ-
ential splicing of the RNA. To our surprise, we

found that knockout of the á-gene in addition tothe â-genes reverts the phenotype. This suggeststhat á-NAC, when not associated with a â-subunit,is toxic to the organism. This conclusion is sup-ported by our finding that wild-type cells overex-pressing the á-subunit are also impaired in growthat higher temperatures. The importance of a bal-anced ratio of á- to â-subunits becomes evident in
the downregulation of subunits in mutant strains
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Figure 7. The absence of NAC does not cause precursor protein accumulation. Log-phasecells of the indicated strains were homogenized after zymolyase treatment (cell lysate),intact cells and nuclei were removed (3000#g sup), mitochondria were pelleted and theremaining supernatant was taken as a cytosolic fraction. An immunoblot was probed withantibodies against HSP60 and YDJ1.

Figure 8. The amount of mistranslocated signalless invertase is negligible in NAC-mutants and WT-cells in vivo. WT- andmutant-strains transformed with plasmids encoding WT-invertase (WT-Inv) or signalless invertase (ÄSS-Inv) were grown tolog-phase on a synthetic complete medium to maintain the plasmids. Starting with an equal cell number 5 ìl of consecutive 10-folddilution steps were spotted onto YPsucrose plates containing bromothymol blue as a pH indicator. Growth to colonies and changeof colour from blue/green to yellow indicates the ability to use sucrose as the only carbon source, e.g. secretion of invertase.

missing other components of the complex. Also,there is more â3-protein expressed from the sameinducible plasmid in the Äâ -mutant than in wild
1

type-cells under the same growth conditions, andit is almost impossible to detect â3-protein in aÄá-strain transformed with the same plasmid.

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The subunits always co-fractionate. We neverobserved single subunits or a shift of one subunitagainst the other(s). The molecular weight of theNAC-containing complex ranges from ca. 48 tomore then 68 kDa in wild-type, â1-knockout, andâ1-knockout strains overexpressing the â3-subunit.The complex of Äá-cells has a smaller (about48 kDa) molecular weight. The á-subunit of adouble â-knockout strain smears across all lanes.That this is not simply the result of poor separ-ation is clearly demonstrated by the elution profilefor the â1-subunit from the strain missing theá- and â3-subunits and the behaviour of the pro-tein standards. To reduce non-specific interactionswith other cytosolic proteins, we increased the saltconcentration to 500 m KOAc and even per-formed the separation under these conditions. Theresult was still the same. The sum of the molecularweight of all three subunits adds up to 48 kDa,which is about the smallest size of complex wedetected. But the â3-subunit is expressed to a lowerlevel than the other two proteins, raising thequestion of what causes the large size. Whatreplaces a knocked-out subunit? The â-subunitsare not in complex with each other but with theá-subunit, as shown by co-immunoprecipitationand indirectly by the ribosome association exper-iment. It may be that in yeast, additional proteinsinteract with á- and â-subunits. These are unlikelyto be the major chaperones as they are too large.YDJ1 is also eliminated as it was found to eluteafter NAC on a sizing column.

When we isolated NAC from bovine braincytosol as a factor interacting with growingpeptide chains at the ribosome, we realized that theâ-subunit had been described before in the litera-ture as a general transcription factor (BTF3,Zheng et al., 1987, 1990; yeast homologues BTT1,EGD1, Shi et al., 1995; Parthun et al., 1992).However, the results of George et al. (1992) andour results, as well as biochemical studies of Floreset al. (1992), do not support a role for NAC intranscription. First, the high affinity binding of thesubunits in the NAC complex make it unlikelythat the â-subunit would dissociate in order tomigrate into the nucleus and influence transcrip-tion, as proposed previously (Zheng et al., 1987,1990; Parthun et al., 1992). Second, we havenever detected any NAC protein in the nucleus bymeans of cell fractionation or immunofluorescence(data not shown) but the amount present might bebelow the detection limit of the techniques weused.


The role of NAC in ribosome binding to theER-membrane was recently questioned by Neuhofet al. (1998) and Raden and Gilmore (1998). Thesegroups have used a modified assay system, whereribosome binding is independent of a signalpeptide and SRP. We were able to reproduce thisdata, but by simply increasing the NAC concen-tration to physiological levels we could restorespecificity (Moller et al., 1998, in press).

Recently, results were published on the partici-pation of NAC in the targeting of mitochondrialproteins (George et al., 1998). Yeast mutants lack-ing both the á-subunit of the NAC and the MFT52protein showed synthetic mitochondrial defects:reduced targeting of reporter proteins, tendency tolose mitochondrial DNA, and changes to organellemorphology. The authors conclude that NAC isinvolved in protein targeting to many (if not all)subcellular locations. It might be difficult to dem-onstrate NACs function in the targeting of pro-teins to the ER in vivo. The fact that the tripleNAC knockout is still viable and the failure of ourinvertase experiment can be explained by the exist-ence of an as-yet unknown substituting factor.Alternatively, NAC may serve a primarily regulat-ory function which is not absolutely necessary forthe cell. Additional proofreading events exist likethe reported opening of the translocon pore by asignal sequence alone (Jungnickel and Rapoport,1995). They ensure that mistargeted peptides willnot be translocated into the ER-lumen. This mech-anism is especially important in yeast, since there alot of proteins are translocated posttranslationally.

We have demonstrated here that yeast NAC is inclose proximity to nascent polypeptides, indepen-dent of whether or not they bear a signal sequence,which resembles the situation in mammaliancytosol. Presently, work is in progress to testwhether yeast NAC can prevent the targeting ofnon-secretory yeast proteins to yeast microsomes.

AbbreviationsNAC, nascent polypeptide-associated complex;

RNC, ribosome nascent polypeptide complex;CAT, chloramphenicol acetyltransferase; KOAc,potassium acetate; ER, endoplasmic reticulum;SRP, signal recognition particle; HSP, heat shockprotein

ACKNOWLEDGEMENTS

We are grateful to H. Ronne (Upsala, Sweden) for
the original knockout strains. Thanks to the
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generous contributors of plasmids and anti-bodies (C. Unger, Berlin; T. Langer, Munich;J. R. Warner, New York; E. Craig, Madison;K. R. Yamamoto, San Francisco; D. Botstein;Cambridge). We greatly appreciated the discussionwith members of the Wiedmann- and Hartmann-laboratories. Special thanks to I. Krenz andE. Burger for excellent technical assistance andto D. Smith who transformed our writing.

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Documents

Initial characterization of the nascent polypeptide-associated complex in yeast