Differential fates of invertase mutants in the yeast endoplasmic reticulum

Differential fates of invertase mutants in the yeastendoplasmic reticulum

ARDYTHE A. McCRACKEN1*, ERIC D. WERNER1, MARGUERITE J. POWELL1,KRISTINA B. KRUSE1 AND JEFFREY L. BRODSKY2

1 Biology Department, University of Nevada, Reno, NV 89557, USA2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

A number of proteins have been identi®ed as substrates for endoplasmic reticulum (ER)-associated proteindegradation (ERAD) and we describe here a new model substrate with which to study this process. Two secretion-defective forms of yeast invertase that accumulated in the ER to greatly different levels were examined: Suc2-538plevels were low, while Suc2-533p was present in high amounts. Because Suc2-533p and Suc2-538p mRNA levelswere comparable, we examined whether Suc2-538p was targeted for degradation. Both mutant polypeptidelevels were unaffected in a yeast strain de®cient in vacuolar protease activity and, additionally, we showed thatSuc2-538p was stabilized in ERAD-de®cient strains, demonstrating that Suc2-538p was a substrate for ERAD.Copyright # 2000 John Wiley & Sons, Ltd.

KEY WORDS Ð ERAD; endoplasmic reticulum-associated protein degradation; endoplasmic reticulum; invertase;chicken antibody

INTRODUCTION

Endoplasmic reticulum (ER)-associated proteindegradation (ERAD) acts as a protein qualitycontrol process by selectively degrading aberrantand unassembled secretory proteins that accumu-late within the ER (for recent reviews see Brodskyand McCracken, 1999; Bonifacino and Weissman,1998). Two reporter proteins used in this labora-tory to study ERAD are the unglycosylated formof the yeast mating pheromone, pro-alpha factor,and the Z variant of human alpha-1 proteaseinhibitor. Studies with these and other yeastproteins have demonstrated that ERAD is ahighly selective quality control process in S.cerevisiae, involving identi®cation and selectionof proteins in the ER, retro-translocation of theseproteins to the cytosol, and subsequent degrada-tion by the proteasome complex (McCracken andBrodsky, 1996; Werner et al., 1996; Hiller et al.,1996; Hampton et al., 1996; Biederer et al., 1996).The mechanism by which ERAD substrates are

selected is not de®ned, and would bene®t greatlyby the identi®cation of new substrates. To thisend, two mutant forms of invertase were studied.

Invertase is a secreted protein that is modi®ed by14 core oligosaccharides in the ER, and outer chainglycosylation in the Golgi apparatus (Taussig andCarlson, 1983; Kaiser and Botstein, 1986). Becauseexpression can be regulated and its activitymeasured easily, invertase makes an excellentreporter protein. We examined invertase mutantsthat lacked the processing indicative of transport tothe Golgi apparatus (Preuss et al., 1991). One,Suc2-533p, has a single amino acid substitution ofa lysine for a glutamate at position 486. This pro-tein is retained in the ER but is not rapidly de-graded. The other, Suc2-538p, is truncated by 119amino acids and lacks one core glycosylation site.This protein is retained in the ER and is degraded,indicating that it is a substrate for ERAD.

MATERIALS AND METHODS

Materials

Invertase was purchased from Sigma. Endogly-cosidase H was purchased from Boehringer-

*Correspondence to: A. A. McCracken, Biology Department ±MS314, University of Nevada, Reno, NV 89557, USA. E-mail:[email protected]

YEAST

Yeast 2000; 16: 49±55.

Received 26 May 1999Accepted 31 August 1999

CCC 0749-503X/2000/010049±07$17.50Copyright # 2000 John Wiley & Sons, Ltd.

Mannheim. Guinea pig and rabbit anti-invertaseantibody were kindly provided by D. Preuss andthe Botstein laboratory (Stanford University) andElizabeth Jones (Carnegie-Mellon University),respectively. Horseradish peroxidase-conjugatedgoat anti-chicken antibody and horseradish per-oxidase-conjugated goat anti-guinea pig antibodywere purchased from Southern Biotechnology.Strains were cultured in medium prepared follow-ing Sherman et al., (1986). Chickens used wereRhode Island Red. Yeast strains and plasmids arelisted in Table 1.

Construction of EWY007

Standard procedures and medium were used forconstruction of the EWY007 strain (Shermanet al., 1986). Yeast AB122 and DBY2449 weremated and prototrophic diploid colonies wereisolated on selection medium, starved for nitrogen,and spores were isolated using a micro-manipulator. Auxotrophic markers were followedby replica plating spore colonies onto selectivemedium, and the mating type of each segregant wasdetermined by crossing to mating-type tester strains

DC14 (MATa his 1) and DC17 (MATa his 1).Overlay tests for protease B and carboxypeptidaseY activity were conducted as described (Jones1991).

Production and isolation of chicken IgY

Invertase was treated with endoglycosidase Haccording to manufacturer's instructions. Degly-cosylated invertase was mixed with Freund'sadjuvant and injected intramuscularly with abooster (without adjuvant) 7 days later. Eggswere collected daily and stored at x20uC. IgY wasisolated from eggs as described (Polson et al.,1980) with some modi®cation. Intact egg yolkswere separated from the albumin by two washeswith distilled H2O and were suspended in two yolkvolumes of buffer (0.01 M PBS, pH 7.5, 0.1 M

NaCl, 0.01% NaN3). Polyethylene glycol 8000(Sigma) was added to a concentration of 3.5% (w/v) for 30 min at room temperature. Samples werethen centrifuged for 10 min at 14 000rg, super-natants were ®ltered through absorbent cotton,brought to 12% (w/v) PEG 8000, vortexed, andcentrifuged for 10 min at 4uC at 14 000rg. The

Table 1. Yeast strains and plasmids.

Strain Genotype Source/reference

DBY2449 MATa ade2-101 ura3-52 suc2-D9 M. Carlson, Columbia UniversityEWY2433 MATa ade2-101 ura3-52 suc2-D9 (pDP169) This laboratoryEWY2438 MATa ade2-101 ura3-52 suc2-D9 (pDP171) This laboratoryEWY2400 MATa ade2-101 ura3-52 suc2-D9 (RB307) This laboratoryAB122 MATa prc1-407 prb1-1122 pep4-3 leu2 ura3-52 A. Brake (Chiron, CA)EWY007 MATa ade2-101 ura3-52 suc2-D9 prcl-407 prb1-1122 pep4-3 This LaboratoryEWY033 MATa ade2-101 ura3-52 suc2-D9 prcl-407 prb1-1122 pep4-3

(pDP169)This Laboratory

EWY038 MATa ade2-101 ura3-52 suc2-D9 prcl-407 prb1-1122 pep4-3(pDP171)

This Laboratory

EWY000 MATa ade2-101 ura3-52 suc2-D9 prc1-407 prb1-1122 pep4-3(RB307)

This Laboratory

BC212 MATa ade2-101 leu2-3-112 ura3-52 his3-D1 B. Courchesne, University of NevadaIK10 MATa ade2-101 leu2-3-112 ura3-52 his3-D1 add7-1 McCracken et al., 1996IK13 MATa ade2-101 leu2-3-112 ura3-52 his3-D1 add3-2 McCracken et al., 1996DC14 MATa his 1 B. Courchesne, University of NevadaDC17 MATa his 1 B. Courchesne, University of Nevada

Plasmid Description Source/reference

RB307 Yep420 Ma et al., 1987pDP169 RB307 plasmid (2m origin) with 3 kb fragment encoding

suc2-533Pruess et al., 1991

pDP171 RB307 plasmid (2m origin) with 3 kb fragment encodingsuc2-538

Pruess et al., 1991

50 A. A. McCRACKEN ET AL.

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 49±55.

precipitates were resuspended in half of theoriginal yolk volume of buffer containing 20 mM

NaN3 and stored at x70uC.

Af®nity chromatography

De-glycosylated invertase was dialysed against0.1 M MOPS, pH 7.5, applied to a 1 ml Af®-gelcolumn, prepared following the manufacturer'sinstructions (BioRad Laboratories) and incubatedfor 15 h at 4uC with rocking. The column wasblocked for 15 h at 4uC with 0.1 M Tris, pH 8.0,then washed three times each with 10 ml of (a)0.1 M acetate, pH 4.0 with 1 M NaCl; (b) 0.1 M

Tris, pH 8.0 with 0.5 M NaCl; and ®nally (c) 0.1 M

MOPS, pH 7.5. The chicken IgY (10 ml) wasadded to the column and incubated for 15 h at 4uCwith rocking. The column was washed with 10 ml10 mM Tris, pH 7.5, containing 0.5 M NaCl, andthe anti-invertase IgY was eluted by threeapplications of a 10 min incubation with 1 ml100 mM glycine, pH 2.5, and each fraction wasimmediately neutralized with 100 ml 1 M Tris,pH 8. The column was washed with 10 mM Tris,pH 8.8, and the ®rst 1 ml fraction was combinedwith the eluted fractions. IgY was concentrated byammonium sulphate precipitation (50%) andstored in PBS with 0.02% NaN3.

Enzyme-linked immunosorbent assay (ELISA)and Western analyses

Yeast lysates were prepared for ELISA analysesfrom 25 OD600 units of cells washed in 10 mM

sodium azide, pelleted and resuspended in 20 mM

Hepes, pH 7.5, 40 mM sorbitol, 150 mM potassiumacetate, 0.5 mM ethylene glycol-bis(b-aminoethylether) N,N,N,N-tetra-acetic acid (EGTA), 2 mM

magnesium chloride, 0.5 M phenylmethylsulphonyl¯uoride (PMSF), 0.1 mM N-tosyl-1-phenylalaninechloromethyl keytone (TPCK), 1 mM benzamidineHCL, 25 mM pepstatin A, and 1 mg/ml lyticase(Sigma) at 37uC for 2 h. Triton X-100 was addedto a ®nal concentration of 1% and samples wereincubated on ice for a minimum of 1 h. Lysateswere centrifuged at 10 000rg for 5 min at 4uCand the supernatant was collected and diluted 1 : 1with protease inhibitor cocktail (32 mM Hepes,pH 7.6, 10 mM ethylenediamine tetra-acetic acid(EDTA), 0.5 mM PMSF, 0.1 mM TPCK, 1 mM

benzamidine HCL and 25 mM pepstatin A).ELISA was performed in triplicate, as previouslydescribed (McCracken et al., 1989), using guineapig anti-invertase antibody.

Yeast lysates were prepared for Western ana-lyses from two OD600 units of cells washed with10 mM NaN3 and stored at x20uC. Cell pelletswere resuspended in 20 ml sample buffer (80 mM

Tris, pH 6.8, 2% SDS, 0.1% bromophenol blue,100 mM DTT, 10% glycerol) and placed in a 95uChot block for 2 min. Then 0.12 g glass beads(450±600 mm, Sigma) were added, and cells weredisrupted on a vortex for 1 min. An 80 ml aliquotof sample buffer was added and samples wereheated at 95uC for 4 min. Proteins were separatedon 7% SDS±PAGE and transferred by electro-phoresis to nitrocellulose membranes (AmershamHybond-C Pure). The membranes were incubatedwith chicken anti-invertase antibody (1 : 1000) for1 h at room temperature with gentle shaking, thenwashed ®ve times with TBS±Tween (Tris-bufferedsaline containing 0.2% Tween 20). Membraneswere incubated with horseradish peroxidase con-jugated goat anti-chicken at 1 : 4000 dilution inblocking solution for 1 h at room temperaturewith gentle shaking, followed by ®ve washes inTBS±Tween and two washes in 50 mM Tris,pH 7.4. The blots were then treated with DABsubstrate (6 mg/ml diaminobezidine, 50 mM Tris,pH 7.4, 0.03% NiCl2, 0.1% H2O2). Membraneswere scanned using a Hewlett-Packard Scan Jet 4cand quanti®ed using BioRad Molecular Analystsoftware.

Immunoblot analyses

Nitrocellulose membrane (Amersham Hybond-C Extra) was wetted in TBS containing 1 mM

MgCl for 30 min and placed in the slot±blotapparatus (BioRad Labs, Richmond, CA). Inver-tase standard was applied to nitrocellulose undervacuum and the membrane was air-dried for30 min, then blocked in TBS containing 3% BSAand 0.2% Tween 20. Membranes were treated andanalysed as for Western analysis.

Immuno-precipitation and protein radiolabelling

Yeast grown in repression medium (5% glucose)were switched to derepression medium (2% lacticacid, pH 5.7) lacking sulphate, and were labelledwith 35S-methionine as described (McCrackenet al., 1989). Radiolabelled invertase was immuno-precipitated using a `sandwich' antibody complexcontaining ®xed Staphylococcus aureus, ®rstbound to rabbit anti-IgY and then bound tochicken anti-invertase. Pulse-chase radiolabellingusing rabbit anti-invertase antibodies and immuno-

TRUNCATED INVERTASE IS AN ERAD SUBSTRATE 51


precipitations were performed as described(Brodsky et al., 1998). Proteins were resolved by10% SDS±PAGE and visualized using a BioRadPhosphoImager.

Northern analyses

Whole cell RNA, puri®ed from cells cultured inderepression medium (2% lactate, pH 5.7) for 7 hto induce the expression of invertase, was analysedby Northern blotting following standard proce-dures (Sambrook et al., 1989). Probes were DNArestriction fragments prepared by random primingaccording to the manufacturer's instructions(United States Biological). Each blot was probed®rst with a 32P-labelled HindIII restriction frag-ment containing 1100 bp of the coding region ofthe SUC2 gene, exposed to ®lm, and then washedand probed a second time with a 32P-labelledHindIII restriction fragment containing 429 bp ofthe TRP1 gene. Autoradiograms were analysed bydensitometry using a Hoeffer GS300 scanningdensitometer.

RESULTS

Because the two secretion-defective forms of yeastinvertase, Suc2-533p and Suc2-538p, accumulatein the ER to greatly different levels (Preuss et al.,1991), it was likely that one was either rapidlydegraded or expressed at low levels. To distinguishbetween these two possibilities, invertase mRNAlevels were analysed in DBY2449 cells containinga deletion of the SUC2 gene and expressing eitherSuc2±533p or Suc2±538p from a 2m plasmid. Cellsexpressing either form of invertase showed com-parable levels of invertase mRNA, while nodetectable invertase RNA was observed in cellsharbouring the plasmid vector without an inver-tase gene insert (Figure 1).

It has been known for some time that yeastsecretory proteins unable to exit the ER may betargeted to ERAD, a proteolytic process indepen-dent of vacuolar protease activity (McCrackenand Kruse, 1993; Finger et al., 1993). Todetermine whether Suc2-538p was a substrate forERAD, both Suc2-533p and Suc2-538p wereexpressed in yeast lacking vacuolar proteases andthe SUC2 gene (EWY033 and EWY038, respec-tively), and a strain lacking SUC2 but containingvacuolar proteases (EWY2433 and EWY2438,respectively). The levels of invertase, determinedby ELISA (Table 2), were the same in both strains,

despite the presence or absence of the vacuolarproteases, indicating that degradation of Suc2-538p did not require transport from the ER to thevacuole. To study the turnover rates of Suc2-533pand Suc2-538p, pulse-chase experiments wereperformed with the EWY033 and EWY038strains. We found that 75% of Suc2-533p butonly 58% of Suc2-538p remained after a 30 minchase (Figure 2). Together, these results indicatedthat Suc2-533p and Suc2-538p are degraded atdifferent rates by a process independent ofvacuolar proteases.

To con®rm that Suc2-538p was a bona ®dereporter protein to study ERAD, Suc2-538p wasexpressed in two ERAD-de®cient yeast strains,IK10 (add7-1) and IK13 (add3-2) (McCrackenet al., 1996), and the levels of invertase were

Figure 1. Levels of invertase mRNA are similar in cellsexpressing suc2-533 or suc2-538. Northern analyses of RNAisolated from yeast transformed with RB307 lacking aninvertase gene insert (EWY2400; lane 1), pDP171 carryingsuc2-538 (EWY2438; lane 2) or pDP169 carrying suc2-533(EWY2433; lane 3). The blot was processed as described (seeMaterials and Methods) using both invertase (Inv) and Trp1radiolabelled probes. The volume density of each band wasdetermined using BioRad Molecular Analysis software. Theratio of Inv to Trp1 signal was similar for the Suc2-533pexpressing cells (r=1.49) compared to the Suc2-538p expres-sing cells (r=1.63).

Table 2. Degradation of truncated invertase is inde-pendent of vacuolar proteases.

Levels of invertase* Suc2-533pStrain (mg/OD of cells) Suc2-538p

EWY033 58.4 5.3EWY038 11.0EWY2433 28.2 5.5EWY2438 5.1

*ELISA assays were performed using guinea-pig anti-invertaseantibody. Values are averages of three ELISA experimentsperformed on cell lysate of cultures depressed in 2% lactic acid.The SD was typically <25% of the mean.



determined by ELISA (Table 3). We found thatSuc2-538p was stabilized in the ERAD mutantscompared to the wild-type parent strain, BC212.

In order to exploit this reporter protein towarda better understanding of the mechanisms of

ERAD, an ample supply of invertase-speci®cantibody was needed. Because the external carbo-hydrates on invertase are highly immunogenic andanti-invertase antibodies often cross-react withmany glycosylated yeast proteins, and sincerabbits often display antibodies to yeast glycopro-teins without immunization, we chose to employchickens as an alternative animal for the produc-tion of antibodies to yeast invertase. Chickens areinexpensive to keep and hens transfer antibodiesfrom the serum to the egg yolk, providing a richsource of immunoglobulin that is easily accessiblethrough non-invasive means (Polson et al., 1980;Gassman et al., 1990).

The titre of anti-invertase chicken IgY, mea-sured by immunoblot analyses (see Materials andMethods), was shown to be quite high throughoutthe collection period, and a peak titre was reachedapproximately 30 days following the initial injec-tion (data not shown). The polyclonal IgY waspuri®ed by immunoaf®nity chromatography toproduce antibody that was highly speci®c forinvertase. To test the speci®city of the puri®edantibody, Western analysis was performed onlysates of EWY033 cultures (expressing Suc2-533p) collected at various times after derepressionof invertase. The results showed that the af®nity-puri®ed IgY was highly speci®c for invertase, sinceother yeast proteins in the cell lysate were notdetected (Figure 3). As expected, we found thatmature invertase migrated in the 102±110 kDarange and the various forms of deglycosylatedinvertase ran below 80 kDa (Preuss et al., 1991),while Suc2-533p migrated at approximately95 kDa, a size consistent with a full-lengthinvertase molecule with core carbohydratesattached, indicating ER accumulation.

Because IgY binds poorly to protein A orprotein G (Harlow and Lane, 1998), we devised an

Table 3. Stabilization of Suc2-538p in ERAD-de®cient mutants.

Levels of Suc2-538p* Mutant/Strain (mg/OD of cells) wild-type

BC212 (wild-type) 3.53IK10 (add7-1) 9.53 2.7IK13 (add3-2) 7.30 2.1

*ELISA assays were performed using guinea-pig anti-invertaseantibody. Values are averages of at least three ELISAexperiments performed on cell lysate of cultures derepressedin 2% lactic acid. The SD was typically <25% of the mean.

Figure 2. Pulse-chase analyses of Suc2-533p and Suc2-538pdegradation. EWY033 and EWY038 cells were metabolicallylabelled with 35S-methionine for 10 min, cycloheximide wasadded, and chase samples were collected at 0, 15, and 30 min(see Methods and Materials). Suc2-533p and Suc2-538p wereimmunoprecipitated from cell extracts using rabbit anti-invertase antibody and protein A±sepharose, and resolved on10% SDS±PAGE. Relative amounts of Suc2-533p and Suc2-538p were determined using a Fuji PhosphorImager andMacBas 2.4 software (Fuji Photo Film. Co. Ltd.).

Figure 3. Af®nity-puri®ed chicken IgY is speci®c for invertase. Af®nity-puri®ed antibody was used in a Western analysisperformed on EWY033 cell lysates prepared at the indicated times (0±18 h) following derepression of the suc2-533 gene by growthin medium containing 2% lactic acid. Standard lanes contain mature (M) or endoglycosidase H-treated invertase (E) added toeither buffer (B) or cell lysate (L). Mature invertase (M-inv) and deglycosylated forms of invertase (©G-inv) are indicated.



antibody `sandwich' (containing ®xed Staphylo-coccus aureus bound subsequently to rabbit-anti-IgY and then chicken anti-invertase) to immuno-precipitate invertase from cell lysates. We foundthat the antibody sandwich ef®ciently capturedendoglycosidase H-treated invertase out of bothbuffer and cell lysate (data not shown). Further-more, when this method was used with radio-labelled EWY033 cells, Suc2-533p was ef®cientlyimmunoprecipitated (Figure 4). Importantly, theimmunoreactive protein was sensitive to endogly-cosidase H treatment and the deglycosylated formmigrated at the predicted 73 kDa size.

DISCUSSION

We describe here two mutant forms of yeastinvertase (Suc2p) that fail to transit through thesecretory pathway, one of which is ef®cientlytargeted for ERAD and one that is not. Both suc2-533 and suc2-538 mRNA levels were comparable(Figure 1), yet the intracellular accumulation ofSuc2-533p was ®ve-fold greater than Suc2-538punder steady-state conditions (Table 2) and Suc2-538p was degraded more rapidly than Suc2-533p(Figure 2). In addition, the electrophoretic migra-tion of Suc2-533p indicated a full-length invertasepolypeptide containing core carbohydrates (Fig-ures 3 and 4), con®rming its accumulation in theER. In contrast, Suc2-538p, a truncated protein,was targeted for degradation by a mechanismindependent of vacuolar proteases (Table 2 andFigure 2) and dependent on the function ofERAD-speci®c mutants (Table 3).

Michaelis and colleagues (Loayza et al., 1998)previously reported that mutant forms of the multi-

spanning membrane protein Ste6p, the a-factortransporter in S. cerevisiae, were retained in the ERbut targeted to differential fates, one stable and onerapidly degraded. Their results suggested theexistence of multiple pathways and checkpointsfor ER protein quality control. Our ®ndingsindicate that two mutant forms of secretedinvertase can be classi®ed similarly and mayprovide a complementary approach to elucidatemechanisms of protein quality control in the ER.Furthermore, comparisons of several such proteinsmay provide clues to understanding the signals fortargeting proteins to the ERAD pathway.

Finally, the chicken anti-invertase IgY con-structed for this project was found to be highlyspeci®c, and techniques for use of the IgY inimmunoprecipitation experiments were estab-lished. However, over time the speci®city of thepuri®ed IgY for invertase decreased (data notshown). The reasons for this are currently underinvestigation.

ACKNOWLEDGEMENTS

We thank Craig Setter and McClain Peterson fortechnical assistance, and Drs D. Botstein, E. Jonesand D. Preuss for plasmids and antibodies.Supported by NSF Grant MCB-9722889.

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Figure 4. Ef®cient immunoprecipitation of Suc2-533p.EWY000 (lanes 1 and 2) or EWY033 (lanes 3 and 4) labelledwith 35S-methionine, as described (McCracken et al., 1989),were immunoprecipitated using the sandwich antibody com-plex (see Materials and Methods). Samples in lanes 2 and 4were treated with endoglycosidase H. *Indicates deglycosylatedSuc2-533p.



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Differential fates of invertase mutants in the yeast endoplasmic reticulum