Copyright C Munksgaard 2001Traffic 2001; 2: 698–704

Munksgaard International Publishers ISSN 1398-9219

Regulation of Organelle Membrane Fusion by Pkc1p

Andrew Lin1, Sheetal Patel1 andMartin Latterich1,2,*

1The Salk Institute, 10010N. Torrey Pine Road, La Jolla, CA

92037, USA2Diversa Corporation, 4955 Directors Place, San Diego, CA

92121, USA

*Corresponding author: Martin Latterich,

mlatterich@diversa.com

Membrane fusion relies on complex protein machiner-ies, which act in sequence to catalyze the fusionof bilayers. The fusion of endoplasmic reticulummembranes requires the t-SNARE Ufe1p, and the AAAATPase p97/Cdc48p. While the mechanisms of mem-brane fusion events have begun to emerge, little isknown about how this fusion process is regulated. Weprovide first evidence that endoplasmic reticulummembrane fusion in yeast is regulated by the action ofprotein kinase C. Specifically, Pkc1p kinase activity isneeded to protect the fusion machinery from ubiquitin-mediated degradation.

Key words: AAA protein, Cdc48, membrane fusion,phosphorylation, protein kinase C, proteolysis, SNARE,Ubc7, ubiquitination, Ufe1

Received 10 July 2001, revised and accepted for publi-cation 26 July 2001

The homotypic fusion of endoplasmic reticulum (ER) mem-branes is a process essential for normal cell division andmaintaining the organelle. In the yeast S. cerevisiae, mem-brane fusion of the ER and contiguous nuclear envelopemust occur during cell division, during nuclear fusion aftermating (karyogamy), and for constitutive maintenance of theER. Although membrane fusion is a process essential formany cellular functions, the detailed series of events bywhich membranes fuse is still not understood.

Studies of the heterotypic membrane fusion of transport ves-icles with their target membranes have identified some of theproteins involved in this process. The soluble N-ethylmaleimi-de sensitive factor (NSF), a member of the AAA family ofATPases, was found to be necessary for fusion of Golgi ves-icles in vitro (1). The yeast homolog, Sec18p, was found in ascreen for mutants deficient in secretion (2). Another cytosol-ic protein, aSNAP (Sec17p in yeast), recruits NSF to mem-branes through its binding to SNAREs (SNAP receptors),forming a 20S complex (3). SNAREs are type II transmem-brane proteins that are capable of forming coiled-coil struc-tures. The discovery that certain SNAREs localized to vesicles(synaptobrevin), while others localized to the plasma mem-brane (syntaxin) (4) led to the SNARE hypothesis. The

698

SNARE hypothesis states that vesicles contain v-SNAREsthat bind to t-SNAREs on the target membrane, conferringspecificity on the process. aSNAP recruits NSF to the SNAREcomplex, which then hydrolyzes ATP, breaking apart theSNARE complex (5). It is believed that this disassembly oc-curs either before membrane fusion, priming the SNAREs forinteraction, or afterwards, resetting the machinery for sub-sequent rounds of fusion.

Previous studies of in vitro reconstituted ER membrane fusionassays have indicated that homotypic fusion of ER mem-branes differs in its protein requirement from the heterotypicfusion of transport vesicles to their target membranes. Homo-typic membrane fusion of organelles does not requireSec18p or Sec17p (6). Instead it depends on a different NSFhomolog, Cdc48p (7), and the ER resident t-SNARE, Ufe1p(8). Fusion is thought to occur after Ufe1p forms a t-t-SNAREpair rather than a v-t-SNARE complex because Ufe1p is es-sential for ER fusion, but no known v-SNAREs are requiredfor the process (8).

While the mechanisms of membrane fusion have begun toemerge, little is known about how these processes are regu-lated. Some recent compelling evidence suggests that phos-phorylation may play a role in controlling membrane fusionevents. Protein phosphatase 1 has been found to be requiredfor a late step in homotypic vacuolar membrane fusion (9).Shp1p, a protein that was found in a screen for mutants thatcompensate for the lethality of an overexpression of proteinphosphatase 1, has been found to bind the known compo-nents of the ER fusion machinery (10). Furthermore, phos-phorylation of a tyrosine residue of Cdc48p has been foundto affect its localization (11), although the biological signifi-cance of this event is not understood.

In addition to phosphorylation, there is evidence suggestingprotein degradation via the ubiquitin/proteasome pathway isconnected to ER membrane fusion. Three proteins that werefound in a screen for proteins that stabilize a ubiquitin-b-galfusion protein (ubiquitin fusion degradation), Ufd1p, Ufd2p,and Ufd3p, were found to interact with Cdc48p (12–14).Ufd1p is of unknown biochemical function, but is known tobe involved in the proteolytic processing of ER membrane-bound transcription factors. The E4 multiubiquitinating ligaseUfd2p binds to ubiquitin moieties of preformed conjugatesand catalyzes the formation of ubiquitin chains. Ufd3p, whichis of unknown biochemical function, is required to maintainproper levels of free Ub. Furthermore, Cdc48p has been iden-tified as a proteasome-interacting protein that exhibits ATP-sensitive association with the 19S cap (15).

Here we show that protein kinase C is required for homotypicmembrane fusion of the endoplasmic reticulum. The yeast

Membrane Fusion and Pkc1p

genome contains a single isoform of protein kinase C, PKC1

(16). The essential yeast Pkc1p is a ser/thr kinase involved in avariety of cellular processes, including cell-wall maintenance,mating, cell-cycle progression, and nutrient sensing (17). Ourresults show that protein kinase C is essential for proper local-ization of Cdc48p, and maintains Ufe1p levels by preventingprotein degradation. Furthermore, the Ufe1p degradation de-fect of pkc1–2 mutants can be compensated for by inactivat-ing the E2 ubiquitin-conjugating enzyme Ubc7p. This regula-tory mechanism establishes a new signaling paradigm ofhow kinase activity controls membrane fusion and the turn-over of the membrane fusion machinery.

Results

Protein kinase C 1 is required for ER membrane

fusion

To address whether phosphorylation plays a role in ER mem-brane fusion, a library of kinase inhibitors were screened fortheir ability to inhibit membrane fusion in an in vitro ER mem-brane fusion assay. The fusion assay is performed by mixingER microsomes from strains that either lack or contain a func-tional ER lumenal glucosidase, GLS1. The glucosidase is re-sponsible for initiating the deglucosylation of newly syn-thesized glycoproteins. Radiolabeled yeast prepro-a-factortranslocated into the lumen of glucosidase-deficient ER(gls1–1 donor membrane) is processed to the deglucosylat-ed form only when the donor membrane fuses with GLS1πacceptor membranes, and lumenal contents are able to mix.Previous studies have shown that oligosaccharide trimmingis a direct measure of membrane fusion (6). Screening kinaseinhibitors using this assay revealed that bisindolylmaleimide,a selective competitive inhibitor of protein kinase C (18), sig-nificantly inhibited ER membrane fusion in a dose-dependentmanner (Figure 1A). These results suggested that Pkc1p or aPkc1p-like activity was needed to drive ER membrane fusion.Pkc1p has indeed been found to associate with the ER (19).To test if Pkc1p directly participated in the fusion of ER mem-branes, recombinant Pkc1 protein was added back to thefusion reaction in the presence of bisindolylmaleimide. Ourresults show that exogenously added Pkc1p is indeed ableto rescue the membrane fusion defect of Pkc-1 inhibitor, sug-gesting that bisindolylmaleimide was specifically targetingthe Pkc1p pathway.

To investigate the role of Pkc1p in vivo, a temperature-sensi-tive mutant, pkc1–2, was examined for a defect in ER mem-brane fusion. pkc1–2 contains a Pro1023Leu mutationlocated in the highly conserved kinase domain of PKC1 (20).ER membranes isolated from a pkc1–2 strain grown at thepermissive temperature (24 æC) were found to be fusion com-petent, similar to that of wildtype membranes (Figure 1B).However, upon temperature shift of the pkc1–2 mutant strainto the restrictive temperature (37 æC) for 2h, subsequentlyisolated ER membranes were found to be defective in mem-brane fusion. The membrane fusion defect observed was uni-lateral in that only one membrane (either donor or acceptor)

699Traffic 2001; 2: 698–704

Figure1: Protein kinase C1 is necessary for ER membranefusion. A. ER microsomes were isolated from WT (MLY2228)yeast, and standard in vitro fusion assays were performed as de-scribed. Membrane fusion ability was tested in the presence of50nM and 150nM bisindolylmaleimide (BIM), or with 50nM bisin-dolylmaleimide and the kinase active PKC1pHA fusion protein immu-nocomplex. Reagents were added to the fusion reaction, and fusionwas allowed to occur at 24 æC for 1h. Assays were also performedwith incubation at 0 æC as a negative control. B. Acceptor mem-branes were isolated from WT (MLY2228) and the temperature-sensitive pkc1–2 mutant (MLY2226) yeast strains grown at the per-missive 24 æC or grown at 24 æC with incubation at 37 æC for 2h.Donor membranes were isolated from gls1–1, PKC1π (MLY1601)strains grown at 24 æC. ER membranes were isolated and fusionassays were performed as described. C. Pkc1–2 microsomes werealso tested for membrane fusion ability in the presence of recom-binant PKC1pHA.

Lin et al.

needs to be derived from pkc1–2 mutants grown at the re-strictive temperature to exhibit a fusion defect. The fusiondefect of pkc1–2 ER membranes could also be compen-sated for by the addition of recombinant Pkc1p (Figure 1C),indicating that the defect was directly related to the loss ofPkc1 protein function.

Protein kinase C 1 is required for proper Cdc48p local-

ization and maintaining Ufe1p levels

To assess the role of Pkc1p in membrane fusion, known com-ponents of the ER fusion machinery, such as Cdc48p, andUfe1p, were examined as putative substrates of Pkc1p phos-phorylation. Detergent-solubilized ER microsomes isolatedfrom WT and pkc1–2 strains grown at the permissive andrestrictive temperatures were analyzed for mobility shifts byWestern blotting. Surprisingly, the levels of Cdc48p andUfe1p were significantly reduced in ER membranes isolatedfrom pkc1–2 mutant strain grown at the restrictive tempera-ture for 2h as compared to pkc1–2 grown at the permissivetemperature, or the wildtype strain (Figure 2A). To furtherstudy the effects of the pkc1–2 mutation, total cellular pro-teins extracted from wildtype and pkc1–2 yeast were ana-lyzed. Our previous studies found that Cdc48p partitions inthe soluble as well as membrane-associated pools (7).Cdc48p immunoblots indicate that total cellular levels ofCdc48p are similar in wildtype and pkc1–2 at the permissiveand nonpermissive temperatures (Figure 2B). However, uponshifting pkc1–2–37 æC, Cdc48p no longer associated with ERmembranes. Therefore, overall Cdc48p levels do not indicatedegradation of the protein in response to Pkc1p inactivation,but rather the protein is released from the ER membrane. Incontrast, protein levels of the ER t-SNARE, Ufe1p, were re-duced below levels of detection in total cell extracts and indetergent-solubilized microsomal extracts of pkc1–2.

Pkc1–2 mutation accelerates the degradation of Ufe1p

The decrease in levels of Ufe1p could be caused by eitherthe inhibition of synthesis of Ufe1p, or an increase in the rateof degradation of the protein. To examine how the pkc1–2

mutation was influencing Ufe1p levels, the effects of thePkc1p loss-of-function mutation on transcription and trans-lation were examined. Pkc1p is known to phosphorylate theMAP kinase kinase kinase, Bck1p, which activates a MAPkinase-signaling cascade, which in turn leads to the acti-vation of specific transcription factors (17). Therefore a loss-of-function mutation in PKC1 that was defective in activatingthe MAP kinase-signaling pathway would fail to transcribe ahost of genes. To test whether Pkc1p specifically affectedtranscription of the UFE1 gene, UFE1 mRNA levels weremeasured from WT and pkc1–2 mutants and compared.Northern analysis revealed that levels of UFE1 mRNA werenot diminished in the pkc1–2 strain at the restrictive tempera-ture (Figure 3A). In fact, pkc1–2 grown at 37 æC exhibited anincreased amount of UFE1 mRNA. Perhaps the loss of Ufe1protein in this strain causes an up-regulation of UFE1 tran-scription. Despite the increased amount of UFE1 mRNA,Ufe1 protein was not detected in ER membranes of pkc1–2

shifted to the restrictive temperature.

700 Traffic 2001; 2: 698–704

Figure2: Ufe1p levels are diminished and Cdc48p is mislocal-ized in pkc1–2 mutants. A. ER microsomes were isolated from WTand pkc1–2 strains grown at 24 æC (lanes 1, 3, respectively) or WTand pkc1–2 strains incubated at 37 æC for 2h (lanes 2, 4, respectively)as described. Samples were analyzed by standard Western blottingas described. The anti-Kar2 and anti-Sec61 blots indicate that equiva-lent microsomes were loaded, and sufficient solubilization of themicrosomes occurred. The pkc1–2 mutation affects levels of micro-somal Cdc48p and Ufe1p, while the Kar2 lumenal chaperone and theSec61 translocon remained unaffected. B. Total yeast protein was ex-tracted from WT and pkc1–2 yeast strains grown at 24 æC (lanes 1, 3,respectively) and WT and pkc1–2 were incubated at 37 æC for 2h(lanes 2, 4) as described. Samples were analyzed by standard West-ern blotting with the indicated antibodies.

Pkc1p is also involved in regulating translation of proteins,potentially by regulating the transcription of ribosomal pro-teins and tRNA (20,21). To determine if pkc1–2 affects Ufe1plevels by inhibiting protein synthesis, a cycloheximide chaseassay was performed. If the pkc1–2 mutation was affectingsynthesis of Ufe1p, then chemically inhibiting translation inWT yeast with cycloheximide would have the same result asthe pkc1–2 mutation. Two hours after the addition of cyclo-heximide, Ufe1p levels in WT yeast were comparable to pre-incubation levels (Figure 3B), but 2h of inactivation of Pkc1pby incubating at the restrictive temperature sharply de-creased the levels of Ufe1p. These results indicate that the

Membrane Fusion and Pkc1p

Figure3: pkc1–2 accelerates the rate of degradation ofUfe1p. A. Northern blot analysis was performed to quantitate UFE1mRNA message. mRNA was isolated from WT and pkc1–2 strainsgrown at 24 æC or with incubation at 37 æC for 2h. Samples wereresolved on a formaldehyde-based gel and transcript levels weredetermined with 32P-labeled UFE1 cDNA probes. ACT1 was exam-ined as a load control. B. Cycloheximide chase assay was per-formed to determine the half-life of Ufe1p. WT cells were treatedwith cycloheximide for 0min, 60min and 120min before total yeastprotein was extracted. pkc1–2 was incubated at 37 æC for 0min,60min, and 120min before total yeast protein was extracted. Thesamples were analyzed by Western blotting using anti-Ufe1p as aprobe. Anti-Kar2p was examined as a load control.

pkc1–2 mutation must be decreasing levels of Ufe1p by ac-celerating its rate of degradation.

The loss of Ufe1p in pkc1–2 mutants and the mislocalizationof Cdc48p could be explained in two ways. One explanationpredicts that the ER membrane fusion machinery is com-prised of Ufe1p serving as an ER membrane receptor forCdc48p, which is required to drive the fusion event. UponPkc1p inactivation and subsequent Ufe1p degradation,Cdc48p is released from the ER membrane and as a resultrenders them incompetent for fusion. Alternatively, within thefusion complex, Cdc48p may protect Ufe1p from activedegradation. When pkc1–2 induces Cdc48p mislocalization,possibly due to an aberrant phosphorylation state, Ufe1pmay become susceptible to the degradation machinery andmembrane fusion becomes inhibited.

Pkc1p controls Ufe1p degradation via the E2 ubiquitin

conjugating enzyme, Ubc7p

The ubiquitin/proteasome pathway has emerged as a crucialcellular pathway in the selective degradation of many pro-

701Traffic 2001; 2: 698–704

teins, including ER-resident proteins (22). Ubiquitination bythe ER-associated E2 ubiquitin conjugating enzyme, Ubc7p,seems to play a major role in ER-associated degradation(23,24). To determine whether Ubc7p is involved in Ufe1pdegradation, a pkc1–2, Dubc7 double mutant was con-structed. The pkc1–2, Dubc7 double mutant was tested forER membrane fusogenicity, and degradation of Ufe1p. Theloss of Ubc7p function in conjunction with the pkc1–2 muta-tion was able to partially rescue the ER membrane fusiondefect of pkc1–2 (Figure 4A) as well as the Ufe1p degrada-tion defect (Figure 4B).

Discussion

Membrane fusion is a highly complex series of carefully regu-lated and timed molecular events and switches. The mem-

Figure4: Dubc7 mutation partially rescues membrane fusiondefect, and Ufe1p degradation defect of pkc1–2 allele. A. ERmicrosomes were isolated from pkc1–2 mutants and pkc1–2,

Dubc7 double mutants grown at 24h, or grown at 24 æC and shiftedto 37 æC for 2h. Membrane fusion assays were performed with mu-tant membranes as acceptors, and gls1–1 as donor membranes.B. Total cellular protein was extracted from pkc1–2 mutants grownat 24 æC and 37 æC (lanes 1, 2, respectively) and pkc1–2, Dubc7

double mutants grown at 24 æC and 37 æC (lanes 3, 4, respectively).Samples were analyzed by Western blotting using anti-Ufe1p andanti-Kar2p as a load control.

Lin et al.

brane fusion machinery of the endoplasmic reticulum in yeasthas emerged to be a valid model for other organelle fusionmachineries, such as the Golgi in mammalian cells (25). Ourpresent study firstly suggests that the protein kinase C(Pkc1p) plays an important role of modulating the ER mem-brane fusion activity by controlling the turn-over of compo-nents of the ER fusion machinery. The turn-over process isfacilitated by the proteasome and seems to be initiated most-ly during Pkc1p inactivation.

Using a chemical library of protein kinase inhibitors, we no-ticed that bisindolylmaleimide, a known competitive inhibitorof protein kinase C, exerted a concentration-dependent inhi-bition of ER membrane fusion in vitro. The direct involvementof Pkc1p in ER membrane fusion was confirmed by addingback Pkc1p to the fusion reaction, which led to a restorationof membrane fusion activity. Furthermore, ER membranesderived from the conditionally defective pkc1–2 mutants areconditionally defective in ER membrane fusion, a defect thatcould be compensated by replenishing functional Pkc1 pro-tein. When investigating the electrophoretic mobility ofknown ER membrane fusion proteins, such as Cdc48p (7) orUfe1p (8), for mobility shifts associated with post-transla-tional modifications, we observed that in pkc1–2 mutantmembranes Cdc48p and Ufe1p levels are greatly reduced.Further investigation showed that Cdc48p appears to be re-leased from the membrane to the cytoplasm, when pkc1–2

mutant strains are shifted to the nonpermissive area. This re-lease is coupled to a degradation of the Ufe1p ER fusionSNARE, thus inactivating fusogenic activity of the ER.

The control of membrane fusion by phosphorylation has beendemonstrated before: vacuole assembly in yeast is subject tocontrol by phosphorylation (9). Phosphorylation of SNAREs inmammalian system is a means to control SNARE pairing andultimately membrane fusion activity (26). Along similar lines,the vesicularization of Golgi and subsequent reassembly iscontrolled by kinases (27). Taken together, differential phos-phorylation and de-phosphorylation events are needed toregulate the fusogenicity of the membrane fusion complex.The control of Ufe1p availability through Pkc1p signaling sug-gests that the turnover of essential membrane fusion compo-nents can be regulated. It remains to be seen if all these mech-anisms of modulating fusion activity transmit a global signal tothe membrane fusion machinery, or if these events are part ofan elaborate local scheme to carefully time the sequence ofmolecular interactions, and activities, and in the case of Pkc1p,turn over the organelle SNARE to inactivate fusion when it isno longer needed. This mechanism may be analogous to themechanism employed by neurotoxins, such as botulinus toxinor tetanus toxin, which shut down synaptic vesicle fusion byproteolytically hydrolyzing the SNAREs (28).

Identifying the target of Pkc1p would help to clarify thesequestions. It is possible that Pkc1p is indirectly involved inregulating membrane fusion. In this case, the target of Pkc1pmay be a downstream kinase. Vacuole fusion in Schizosac-

charomyces pombe requires Pmk1p, a homolog of the S.

702 Traffic 2001; 2: 698–704

cerevisiae Mpk1p, which is a MAP kinase that is activated byPkc1p (29). However, recent evidence suggests Pkc1p maybe directly involved in regulating fusion activity, targetingcomponents of the fusion machinery. Human PKC has beenshown to directly phosphorylate the t-SNARE, syntaxin 4, inin vitro experiments (26). Furthermore, the Cdc48 homolog,NSF, is phosphorylated by PKC in rat synaptosomes (30).Another possibility is that some protein that has yet to beidentified as part of the fusion machinery is the target ofPkc1p. Our future studies will address the exact nature ofPkc1p’s involvement in regulating the fusion machinery.

Apart from the biochemical requirement of Cdc48p for ERmembrane fusion, Cdc48p has been linked to a number ofother cellular pathways, most prominently the ubiquitin-de-pendent protein degradation pathway (12–15). While theprecise role for Cdc48p seems to be somewhat obscure be-cause there has not been a demonstrated biochemical activ-ity for Cdc48p in the Ub-dependent pathway, it seems plaus-ible that Cdc48p could function as an unfoldase to disas-semble protein complexes where one component is subjectto degradation or removal. Cdc48p and its mammalian ortho-log p97 share features consistent with unfoldases (31). It isthus possible that Cdc48p remains associated with the ERmembrane fusion complex, until a lack of Pkc1p-dependentphosphorylation event triggers a removal of Ufe1p and tar-geting of Ufe1p for destruction of the proteasome. Cdc48pcould act in the capacity to extract Ufe1p from the mem-brane. Future investigation of this mechanism will provide aview of the precise molecular function of the Cdc48 protein.It is likely that the Ufe1p is degraded through an ubiquitin-dependent step, since deletions in the ubiquitin-conjugatingUBC7 gene prevent proteolysis of Ufe1p in response to lossof Pkc1p function. Future experiments will address if Ufe1pis directly ubiquitinated in this process, and where on theprotein the ubiquitination occurs. ubc7 deletions itself do notaffect ER membrane fusion (M.L., unpublished data) or ves-icular traffic (32); however, it cannot be ruled out that otherpromiscuous ubiquitin conjugating enzymes take over thisrole, should it be essential for SNARE turn-over.

Turnover of proteins is an energetically expensive step, andthe regulated turnover of proteins is usually the result of aspecific requirement to tightly regulate a biochemical mech-anism. The reason the ER membrane fusion SNARE Ufe1pis turned over could be that the protein has a longer half-life,and that the ER membrane fusion and retrograde vesicle fu-sion have to be down-regulated during the cell cycle or cer-tain environmental stimuli through the action of Pkc1p. Alter-natively, Pkc1p when activated through external stimuli, mayprevent the basal turn-over of Ufe1p and enhance ER mem-brane fusion capacity and retrograde traffic in response tothe stimulus. We favor the model in which activated Pkc1pprevents the breakdown of Ufe1p and permits the cell tohave a more active fusion capacity.

In summary, our study suggests that the ER membrane fu-sion machinery is subject to elaborate regulation mechan-

Membrane Fusion and Pkc1p

isms via phosphorylation/dephosphorylation and degradationvia the proteasome. It remains to be seen what detailed se-quence of events trigger and sustain the turn-over of Ufe1pand what the biological need is for this elaborate regulatorystep.

Materials and Methods

Strains and antibodies

Strains used in this study have been described elsewhere (8,33) or wereconstructed using standard yeast genetics. The UBC7 knockout plasmidwas a generous gift from R. Hampton, UCSD. The plasmid encoded theUBC7 gene interrupted with a Leu marker. The plasmid was linearized byrestriction digesting, and transformed into the pkc1–2 strain (MLY2226).Transformation was performed by the LiAc method, and transformantswere selected for on YMM-Leu plates. Antibodies used in this study havebeen described elsewhere (6,8). Rabbit anti-Cdc48p antibodies were agenerous gift of K. Frohlich, Tubingen. Rabbit anti-Sec61p antibodies werea generous gift of R. Schekman, UC Berkeley. Rabbit anti-Kar2p were agenerous gift of Jeff Brodsky. Rabbit anti-Ufe1p antibodies were a gener-ous gift of H. Pelham, Cambridge. Horseradish peroxidase conjugated goatanti-rabbit antibodies were from Amersham Pharmacia (Princeton, NJ,USA). Bisindolylmaleimide and cycloheximide were from Sigma (St Louis,MO, USA). A plasmid containing an HA-tagged Pkc1p was a generous giftof D. Levin, John Hopkins University. The plasmid was transformed to wild-type yeast and expression was induced by growing in galactose-containingmedia (34). Immunoprecipitation was performed as described (35), usingthe 12CA5 monoclonal anti-HA antibody.

ER membrane isolation and membrane fusion assay

The isolation of ER membranes and the ER membrane fusion assay wereperformed as previously described (8).

Western blotting of ER microsomes and whole cell lysates

Standard Western blotting techniques have been described elsewhere(34). ER microsomes isolated from WT and pkc1–2 were washed 2¿with Buffer 88 and were resuspended in reducing protein sample buffercontaining 5% b-mercaptoethanol. Samples were heated at 55 æC for15min prior to analysis by SDS-PAGE. Semi-dry transfer to nitrocellulosewas performed using Bio-Rad’s Western transfer apparatus (Hercules, CA,.USA) at 20V for 42min. Membranes were blocked with 5% nonfat drymilk in phosphate-buffered saline containing 0.05% Triton X-100 (PBS-T). Antibodies were used at dilutions of 1 :5000 and detected by HRP-conjugated secondary antibodies. Blots were developed using AmershamLife Science’s ECL detection reagents. Total cellular proteins were ex-tracted from WT and pkc1–2 yeast strains. Twenty-five milliliters of yeastculture was grown to an OD of 0.5. Cells were harvested at 5000¿gfor 5min. Cells were resuspended in 6ml fresh media, and incubated atpermissive or restrictive temperatures for 2h. Equivalent cells, as deter-mined by A600 were harvested 5000¿g for 5min in a 2-ml Eppendorftube. One hundred microliters of SUME (1% SDS, 8M Urea, 10mM MOPSpH6.8, 10mM EDTA) were added, as were 100ml acid-washed glassbeads. Cells were vortexed 8¿ for 30s. One hundred microliters of reduc-ing protein sample buffer containing 5% b-mercaptoethanol was added.Samples were analyzed by Western blotting, as described with anti-Ufe1pand anti-Cdc48p

Northern blotting

Standard Northern blotting techniques have been described elsewhere(34). RNA was extracted from wildtype and pkc1–2 mutant yeast grownat 24 æC and 37 æC by the SDS/double-phenol extraction method. Poly ARNA was purified with a Qiagen mRNA extraction kit (Valencia, CA, USA).

703Traffic 2001; 2: 698–704

Five micrograms of poly A RNA was resuspended in sample buffer (3.5mlH20, 5ml formamide, 1.5ml MOPS, 2ml 13.2M formaldehyde), heated to65 æC for 5min, and chilled on ice. RNA was resolved on a formaldehyde-based gel (111ml H20, 15ml 10¿ MOPS, 1% agarose, 24ml formalde-hyde) in Running Buffer (74% H20, 10¿ MOPS, 16% formaldehyde). Gelwas prerun at 8V/cm for 10min. Samples were loaded, and gel was runat 5V/cm. Gel was soaked in DEPC H20 for 15s, and transferred to immobilinmembrane by capillary transfer. Membranes were crosslinked at1200 joules¿100. Membranes were prehybridized for 3h at 65 æC in hybridi-zation solution (1.5¿ SSPE, 7% SDS, 250mg/ml heparin, 100mg/ml soni-cated and heat-denatured salmon-sperm DNA) The membranes wereprobed with UFE1 and ACT1 cDNA probes labeled by random primers andadded to hybridization solution. Membranes were hybridized overnight at65 æC, and washed in Wash solution (0.1¿ SSC, 0.1% SDS) at 65 æC for15min. Blots were visualized on a phosphorimager screen, and bands werequantitated with Amersham Pharmacia’s Image Quant (Princeton, NJ, USA).

Cycloheximide chase assay

Cycloheximide chase assay was performed on WT and pkc1–2 yeaststrains as described (36). Twenty-five-microliter yeast cultures were grownto an OD of 0.3, and harvested at 5000¿g for 5 min. After resuspensionin 6ml fresh media, an aliquot was taken as timeΩ0min. Cycloheximidewas added at a dilution of 1 :1000. Cultures were incubated at the restric-tive temperature. At timeΩ2h, and 4h, equivalent cells were taken fromculture and total cellular proteins were extracted as described. Sampleswere then analyzed by Western blotting, as described, using anti-Ufe1pand anti-Kar2p.

Acknowledgments

We would like to thank Susana Prieto, Kendall Powell, Virginia Butel, SureshSubramani, and Senyon Choe for reading the manuscript. We thank An-gelica Diaz, and Irma Padilla for superb technical assistance, and TonyHunter and Randy Hampton for stimulating discussions. This work wassupported by grants from the NIH GM54729 and the Dorsett-Brownfoundation to ML, and NIH training grant T32G 007240 to AL. ML is aPew Scholar.

References

1. Block MR, Glick BS, Wilcox CA, Weiland FT, Rothman JE. Purificationof an N-ethylmaleimide-sensitive protein catalyzing vesicular trans-port. Proc Natl Acad Sci USA 1988;33:7852–7856.

2. Novick P, Field C, Sheckman R. Identification of 23 complementationgroups required for post-translational events in the yeast secretorypathway. Cell 1980;21:205–215.

3. Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H,Geroanos S, Tempst P, Rothman J. SNAP receptors implicated in ves-icle targeting and fusion. Nature 1993;362:318–324.

4. Boudier J, Charvin N, Boudier JL, Fathallah M, Tagaya M, TakahashiM, Seagar MJ. Distribution of components of the SNARE complex inrelation to transmitter release sites at the frog neuromuscular junction.Eur J Neurosci 1996;8:545–552.

5. Rothman JE, Warren G. Implications of the SNARE hypothesis forintracellular membrane topology and dynamics. Curr Biol 1994;4:220–233.

6. Latterich M, Sheckman R. The karyogamy gene KAR2 and novel pro-teins are required for ER-membrane fusion. Cell 1994;78:87–98.

7. Latterich M, Frohlich KU, Sheckman R. Membrane fusion and the cellcycle: Cdc48p participates in the fusion of ER membranes. Cell1995;82:885–893.

8. Patel SK, Indig FE, Olivieri N, Levine ND, Latterich M. Organelle mem-

Lin et al.

brane fusion: a novel function for syntaxin homolog Ufe1p ER mem-brane fusion. Cell 1998;92:611–620.

9. Peters C, Andrews PD, Stark M, Csaro-Tadic S, Glatz A, PodtelejnikovA, Mann M, Mayer A. Control of the terminal step of intracellularmembrane fusion by protein phosphatase 1. Science 1999;285:1084–1087.

10. Zhang S, Guha S, Volkert F. The Saccharomyces SHP1 gene, whichencodes a regulator of phosphoprotein phosphatase 1 with differentialeffects on glycogen metabolism, meiotic differentiation and mitoticcell cycle progression. Mol Cell Biol 1995;15:2037–2050.

11. Madeo F, Schlauer J, Zischka H, Mecke D, Frohlich KU. Tyrosine phos-phorylation regulates cell cycle-dependent nuclear localization ofCdc48p. Mol Biol Cell 1998;9:131–141.

12. Hoppe T, Matuschewski K, Rape M, Schlenker S, Ulrich HD, Jentsch S.Activation of membrane-bound transcription factor by regulated ubiqui-tin/proteasome-dependent processing. Cell 2000;102:577–586.

13. Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S.A novel ubiquitination factor, E4, is involved in multiubiquitin chainassembly. Cell 1999;96:635–644.

14. Ghislain M, Dohmen R, Levy F, Varshavsky A. Cdc48p interacts withUfd3p, a WD repeat protein required for ubiquitin-mediated proteol-ysis in Saccharomyces cerevisiae. EMBO J 1996;15:4884–4899.

15. Verma R, Chen S, Feldman R, Schieltz D, Yates J, Dohmen J, DeshaiesRJ. Proteasomal proteomics. Identification of nucleotide-sensitive pro-teasome-interacting proteins by mass spectrometric analysis of affin-ity-purified proteasomes. Mol Biol Cell 2000;11:3425–3439.

16. Levin DE, Fields FO, Kunisawa R, Bishop JM, Thorner J. A candidateprotein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle.Cell 1990;62:213–224.

17. Heinisch JJ, Lorberg A, Schmitz H, Jacoby JJ. The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellularintegrity in Saccharomyces cerevisiae. Mol Microbiol 1999;32:671–680.

18. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, AjakaneM, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D,Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent andselective inhibitor of protein kinase C. J Biol Chem 1991;266:15771–15781.

19. Park H, Lennarz WJ. Evidence for interaction of yeast protein kinase Cwith several subunits of oligosaccharyl transferase. Glycobiology2000;10:737–744.

20. Levin DE, Bartlett-Heubusch E. Mutants in S. cerevisiae PKC1 genedisplay a cell cycle-specific osmotic stability defect. J Cell Biol 1992;116:1221–1229.

21. Li Y, Moir RD, Sethy-Coraci IK, Warner JR, Willis IM. Repression ofribosome and tRNA synthesis in secretion-defective cells is signaledby a novel branch of the cell integrity pathway. Mol Cell Biol2000;20:3843–3851.

704 Traffic 2001; 2: 698–704

22. Sommer T, Wolf DH. Endoplasmic reticulum degradation. reverse pro-tein flow of no return. FASEB J 1997;11:1227–1233.

23. Hiller MM, Finger A, Schweiger M, Wolf DH. ER degradation of amisfolded lumenal protein by the cytosolic ubiquitin-proteasomepathway. Science 1996;273:1725–1728.

24. Hampton RY, Bhakta H. Ubiquitin-mediated regulation of 3-hydroxy-3 methylglutaryl-CoA reductase. Proc Natl Acad Sci USA 1997;94:12944–12948.

25. Rabouille C, Levine TP, Peters JM, Warren G. An NSF-like ATPase, p97,and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell1995;82:905–914.

26. Chung SH, Polgar J, Reed GL. Protein kinase C phosphorylation ofsyntaxin 4 in thrombin-activated human platelets. J Biol Chem 2000;275:25286–25291.

27. Malhotra V, Warren G. The organization of the Golgi apparatus. CurrOpin Cell Biol 1998;10:493–498.

28. Burns D, Engers S, Yang C, Ossig R, Jeromin A, Jahn R. Inhibition oftransmitter release correlates with the proteolytic activity of tetanustoxin and botulinus toxin A in individual cultured synapses of Hirudo

medicinalis. J Neurosci 1997;17:1898–1910.29. Bone N, Millar JB, Armstrong J. Regulated vacuole fusion and fission

in Schizosaccharomyces pombe: an osmotic response dependent onMAP kinases. Curr Biol 1998;8:135–144.

30. Matveeva EA, Whiteheart SW, Vanaman TC, Slevin JT. Phosphoryla-tion of the N-ethylmaleimide-sensitive factor is associated with depol-arization-dependent neurotransmitter release from synaptosomes. JBiol Chem 2001;276:12174–12181.

31. Patel S, Latterich M. The AAA team: related ATPases with diversefunctions. Trends Cell Biol 1998;8:65–71.

32. Kowlaski JM, Parekh RN, Mao J, Wittrup KD. Protein folding stabilitycan determine the efficiency of escape from endoplasmic reticulumquality control. J Biol Chem 1998;273:19453–19458.

33. Huang KN, Symington LS. Mutation of the gene encoding protein ki-nase C1 stimulates mitotic recombination in Saccharomyces cerevis-

iae. Mol Cell Biol 1994;14:6039–6045.34. Ausbel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA,

Struhl K. Current Protocols in Molecular Biology. Boston: John Wiley &Sons, Inc.; 1995.

35. Watanabe M, Chen C, Levin DE. Saccharomyces cerevisiae PKC1 en-codes a protein kinase C (PKC) homolog with a substrate specificitysimilar to that of mammalian PKC. J Biol Chem 1994;269:16829–16836.

36. Gardner R, Cronin S, Leader B, Rine J, Hampton R, Leder B. Sequencedeterminants for yeast 3-hydroxy-3-methylglutaryl-CoA reductase, anintegral endoplasmic reticulum membrane protein. Mol Biol Cell 1998;9:2611–2626.