The Vacuolar Import and Degradation Pathway Merges with the Endocytic Pathway to Deliver Fructose-1,6-bisphosphatase to the Vacuole for Degradation
2008; Elsevier BV; Volume: 283; Issue: 38 Linguagem: Inglês
10.1074/jbc.m709922200
ISSN1083-351X
AutoresC. Randell Brown, Allison B. Wolfe, Dongying Cui, Hui-Ling Chiang,
Tópico(s)Autophagy in Disease and Therapy
ResumoThe gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the ϵ-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Δvph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway. The gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is degraded in the vacuole when glucose is added to glucose-starved cells. Before it is delivered to the vacuole, however, FBPase is imported into intermediate carriers called Vid (vacuole import and degradation) vesicles. Here, using biochemical and genetic approaches, we identified a requirement for SEC28 in FBPase degradation. SEC28 encodes the ϵ-COP subunit of COPI (coat protein complex I) coatomer proteins. When SEC28 and other coatomer genes were mutated, FBPase degradation was defective and FBPase association with Vid vesicles was impaired. Coatomer proteins were identified as components of Vid vesicles, and they formed a protein complex with a Vid vesicle-specific protein, Vid24p. Furthermore, Vid24p association with Vid vesicles was impaired when coatomer genes were mutated. Kinetic studies indicated that Sec28p traffics to multiple locations. Sec28p was in Vid vesicles, endocytic compartments, and the vacuolar membrane in various mutants that block the FBPase degradation pathway. Sec28p was also found in vesicles adjacent to the vacuolar membrane in the ret2-1 coatomer mutant. We propose that Sec28p resides in Vid vesicles, and these vesicles converge with the endocytic pathway. After fusion, Sec28p is distributed on the vacuolar membrane, where it concentrates on vesicles that pinch off from this organelle. FBPase also utilizes the endocytic pathway for transport to the vacuole, as demonstrated by its presence in endocytic compartments in the Δvph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway. Transport of proteins and lipids between organelles is an important function of all eukaryotic cells. In many cases, vesicles facilitate the transport of cargo proteins or lipids from donor membranes to acceptor membranes (1Owen D.J. Collins B.M. Evans P.R. Annu. Rev. Cell Dev. Biol. 2004; 20: 153-191Crossref PubMed Scopus (361) Google Scholar, 2Owen D.J. Biochem. Soc. 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The most thoroughly studied transporters are coat protein complex I (COPI), 2The abbreviations used are: COPIcoat protein complex IERendoplasmic reticulumFBPasefructose-1,6-bisphosphataseARFADP-ribosylation factorHAhemagglutininDSPdithiobis(succinimidyl propionate)GFPgreen fluorescent proteinMALDImatrix-assisted laser desorption ionizationVidvacuole import and degradation. COPII, and clathrin-coated vesicles. COPI vesicles mediate the retrograde transport from the Golgi to the ER as well as intra-Golgi transport, whereas COPII-coated vesicles conduct anterograde transport from the ER to Golgi. The clathrin-coated vesicles regulate trafficking from the plasma membrane to early endosomes and from the Golgi to endosomes (1Owen D.J. Collins B.M. Evans P.R. Annu. Rev. Cell Dev. Biol. 2004; 20: 153-191Crossref PubMed Scopus (361) Google Scholar, 2Owen D.J. Biochem. Soc. Trans. 2004; 32: 1-14Crossref PubMed Scopus (36) Google Scholar, 3Wieland F. Harter C. Curr. Opin. 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Emr S.D. Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 277-298Crossref PubMed Scopus (453) Google Scholar). For example, a specialized autophagy pathway has been studied in our laboratory. This pathway utilizes a novel type of vesicle called Vid (vacuole import and degradation) vesicles, which transport cargo such as fructose-1,6-bisphosphatase (FBPase). FBPase is a key regulatory enzyme in gluconeogenesis. This enzyme is induced when yeast cells are grown under glucose starvation conditions. However, when glucose-starved cells are shifted to fresh glucose, FBPase is rapidly inactivated and then degraded (27Chiang H.-L. Schekman R. Nature. 1991; 350: 313-318Crossref PubMed Scopus (131) Google Scholar, 28Chiang M.C. Chiang H.-L. J. Cell Biol. 1998; 140: 1347-1356Crossref PubMed Scopus (66) Google Scholar, 29Shieh H.-L. Chiang H.-L. J. Biol. Chem. 1998; 273: 3381-3387Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). The glucose-induced degradation of FBPase has been reported to occur both in the proteasome (31Schork S.M. Bee G. Thumm M. Wolf D.H. FEBS Lett. 1994; 349: 270-274Crossref PubMed Scopus (59) Google Scholar, 32Hammerle M. Bauer J. Rose M. Szallies A. Thumm M. Dusterhus S. Mecke D. Entian K.D. Wolf D.H. J. Biol. Chem. 1998; 273: 25000-25005Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 33Regelmann J. Schule T. Josupeit F.S. Horak J. Rose M. Entian K.D. Thumm M. Wolf D.H. Mol. Biol. Cell. 2003; 14: 1652-1663Crossref PubMed Scopus (122) Google Scholar) and in the vacuole (27Chiang H.-L. Schekman R. Nature. 1991; 350: 313-318Crossref PubMed Scopus (131) Google Scholar, 28Chiang M.C. Chiang H.-L. J. Cell Biol. 1998; 140: 1347-1356Crossref PubMed Scopus (66) Google Scholar, 29Shieh H.-L. Chiang H.-L. J. Biol. 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However, when glucose was added to cells that have been starved for longer periods of time, FBPase was degraded in the vacuole (35Hung G.C. Brown C.R. Wolfe A.B. Liu J. Chiang H.L. J. Biol. Chem. 2004; 279: 49138-49150Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Malate dehydrogenase (MDH2) is another gluconeogenic enzyme that shares degradation characteristics with FBPase. Short-term starvation leads to proteasome-dependent degradation of MDH2, whereas long-term starvation leads to vacuolar degradation of this protein (35Hung G.C. Brown C.R. Wolfe A.B. Liu J. Chiang H.L. J. Biol. Chem. 2004; 279: 49138-49150Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). FBPase is transported to the vacuole via Vid vesicles (36Huang P.H. Chiang H.-L. J. Cell Biol. 1997; 136: 803-810Crossref PubMed Scopus (80) Google Scholar). At present, the origin of these vesicles has not been established, although we have partially characterized these structures. As such, we have determined that FBPase import into Vid vesicles requires the heat shock protein Ssa2p (37Brown C.R. McCann J.A. Chiang H.-L. J. Cell Biol. 2000; 150: 65-76Crossref PubMed Scopus (62) Google Scholar), Vid22p (38Brown C.R. McCann J.A. Hung G. Elco C. Chiang H.-L. J. Cell Sci. 2002; 115: 655-666Crossref PubMed Google Scholar), and cyclophilin A (39Brown C.R. Cui D. Hung G. Chiang H.-L. J. Biol. Chem. 2001; 276: 48017-48026Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Once they are loaded, Vid vesicles are transported to the vacuole through a process that is dependent on the presence of Vid24p, Ypt7p, SNARE proteins, and V-ATPase (40Brown C.R. Liu J. Hung G. Carter D. Cui D. Chiang H.-L. J. Biol. Chem. 2003; 278: 25688-25699Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 41Liu J. Brown C.R. Chiang H.L. Autophagy. 2005; 1: 146-156Crossref PubMed Scopus (10) Google Scholar). As stated above, the origin of Vid vesicles remains unknown. This is largely due to the lack of specific Vid vesicle markers. Vid24p remains the only known marker for these structures to date. Further complicating these studies, Vid24p is present at very low levels prior to a glucose shift. Synthesis of Vid24p does increase following a glucose shift for 20 min, at which point it localizes to Vid vesicles as a peripheral protein (28Chiang M.C. Chiang H.-L. J. Cell Biol. 1998; 140: 1347-1356Crossref PubMed Scopus (66) Google Scholar). In our previous studies, multiple FBPase containing organelles have been observed to accumulate in vid mutants (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar), suggesting that organelles other than Vid vesicles may also be involved in the delivery of FBPase to the vacuole (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). In an attempt to identify additional Vid vesicle marker proteins and track the Vid vesicle trafficking pathway, purified vesicle fractions were subjected to MALDI analysis. Via this method, we identified COPI coatomer proteins including Sec28p, Sec21p, and Ret1p as components of purified Vid vesicles. SEC28, which encodes the ϵ-COP component of coatomers (42Duden R. Kajikawa L. Wuestehube L. Schekman R. EMBO J. 1998; 17: 985-995Crossref PubMed Scopus (67) Google Scholar, 43Kimata Y. Higashio H. Nies A.T. Spring H. Brom M. Keppler D. J. Biol. Chem. 2000; 275: 10655-10660Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), was also identified independently via the screening of a deletion library using a previously established colony blot protocol (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). Here, we show that SEC28 and coatomer genes play a role in the FBPase degradation process. Δsec28 and coatomer mutants exhibited defective FBPase degradation, whereas FBPase association with Vid vesicles was also impaired in these mutant strains. Moreover, coatomer subunits were found in Vid vesicle containing fractions, where they formed a protein complex with Vid24p. Sec28p was also found in Vid vesicles, endosomes, and vacuole membranes in various mutants that block the FBPase degradation pathway. We propose that Sec28p resides on Vid vesicles, and these vesicles later merge with the endocytic pathway. This idea was further confirmed by a study of FBPase distribution in the Δvph1 strain. FBPase was initially in the cytosol but moved to endocytic compartments at later time points. This suggests that FBPase enters the endocytic pathway following a glucose shift. Taken together, our results establish a strong connection between the endocytic pathway and the vacuole import and degradation pathway that delivers cytosolic FBPase to the vacuole for degradation. Yeast Strains, Antibodies, and Primers—S. cerevisiae strains used in this study are listed in Table 1. The deletion strains derived from BY4742 were from Euroscarf (Euroscarf, Germany). The coatomer mutants (ret1-1, ret1-3, sec21-1, and sec27-1) and anti-Sec28p sera were gifts from Dr. Rainer Duden (University of Cambridge, UK). The coatomer mutants (ret2-1, ret3-1, sec21-1, and sec27-1) were gifts from Dr. Howard Riezman (University of Geneva, Switzerland). Anti-coatomer sera were obtained from Dr. R. Schekman (University of California, Berkeley, CA). The rhodamine goat anti-mouse and fluorescine goat anti-rabbit antisera were purchased from Covance. Mouse monoclonal anti-HA was purchased from Roche. The enhanced chemiluminescence kit was purchased from PerkinElmer Life Sciences. Primers used in this study are listed in Table 2.TABLE 1Strains used in this study Deletion strains derived from BY4742 were from Euroscarf.StrainGenotypeHLY635MATα ura3-52 LEU2 trp1Δ63 his3Δ200 GAL2BY4742MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0Δsec28MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 sec28::kanMX4HLY1080MATα leu2Δ0 lys2Δ0 ura3Δ0 FBPase-GFP::HIS3HLY1385MATα leu2Δ0 lys2Δ0 ura3Δ0 sec28::kanMX4 FBPase-GFP::HIS3HLY2119MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 vam3::kanMX4 FBPase-GFP::HIS3HLY225MATa his3-Δ200 ura3-52 leu2,3-112 lys2-801 vid24::TRP1Δvam3MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 vam3::kanMX4HLY1023MATα leu2Δ0 lys2Δ0 ura3Δ0 vam3::kanMX4 VID24-HA::HIS3HLY1816MAT a lys2ΔO ura3ΔO leu2ΔO sec2-1 Vid24-HA::HIS3RSY1010MATα ura3 leu2 sec21-1RH3517MATα ura3 his3 leu2 lys2 suc2Δ9 ret3-1 mycSTE2EMP47tail::URA3RH3521MATa sec27-1 his4 ura3 leu2 bar1 mycSTE2EMP47ptail::URA3RH3516MATα his3 ura3 leu2 lys2 suc2Δ9 ret2-1 mycSTE2EMP47tail::URA3YW05MATα lys1 leu2 ura3 trp1 ubc1::HIS3Δise1MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ise1::kanMX4Δarf1MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 arf1::kanMX4HLY228MATα leu2Δ0 lys2Δ0 ura3Δ0 VID24-HA::HIS3HLY1386MATα leu2Δ0 lys2Δ0 ura3Δ0 sec28::kanMX4 VID24-HA::HIS3HYL1817MATα leu2 ura3 ret2-1 Vid24-HA::HIS3HLY1816MATa leu2 ura3 sec21-1 Vid24-HA::HIS3HLY1422MATα ura3 leu2 lys1 Sec28p-GFP::HIS3Δypt7MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ypt7::kanMX4Δubc1MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ubc1::kanMX4Δvph1MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 vph1::kanMX4HLY1412MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 vam3::kanMX4 Sec28p-GFP::HIS3HLY1463MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 ubc1::kanMX4 Sec28p-GFP::HIS3HLY1793MATα his3 ura3 leu2 lys2 suc2Δ9 ret2-1 mycSTE2EMP47tail::URA3 Sec28p-GFP::HIS3HLY2113MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 vph1::kanMX4 Sec28p-GFP::HIS3HLY1747MATα leu2Δ0 lys2Δ0 ura3Δ0 vph1::kanMX4 FBPase-GFP::HIS3Δpep4MATα leu2Δ0 lys2Δ0 ura3Δ0 pep4::kanMX4W303MATα ura3 leu2 his3 trp1 lys2 ade2 Open table in a new tab TABLE 2Primers used in this studyPrimersFBPase-GFP ForwardATTTGGTTGGGTTCTTCAGGTGAAATTGACAAATTTTTAGACCATATTGGCAAGTCACAGCGGATCCCCGGGTTAATTAA ReverseCCATCCCATTCCATTCGCTACTTCCTTTCTCTTTTCCTAAGAATTTTCATTATTAGAAGGGAATTCGAGCTCGTTTAAACVid24p-HA ForwardCATCTTTGAAAAATAAAGTCGAGTCCAGTGATTGTTCTTTTGAGTTTGCTCGGATCCCCGGGTTAATTAA ReverseTAGACATAGACATGCTGTTATCATACCAAATAGAAAAGTGTACAGTCTTTGAATTCGAGCTCGTTTAAACSec28p-GFP ForwardCACCAAGAAATTGACGCAAAATTCGATGAATTAGTGAGGAAATATGATACGTCCAACCGGATCCCCGGGTTAATTAA ReverseATGAAATATTTTTTTCTTTTTCTAAAAAACCTACATGTTTAATGTGAGATATTACGTAAAGAATTCGAGCTCGTTTAAAC Open table in a new tab FBPase Colony Blot Assay—The colony blot assay was performed as described (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). A yeast deletion library was plated on YPKG plates for 5–7 days at 22 °C, after which mutant strains were replica plated onto nitrocellulose membranes. Nitrocellulose membranes were incubated with affinity purified anti-FBPase antibodies at 1:1000 dilution, washed, and incubated with alkaline phosphatase-conjugated goat anti-rabbit antibodies at 1:5000 dilution. FBPase degradation-deficient mutants were identified as dark purple colonies. SEC28 was identified using this method. Differential Centrifugation, Sucrose Density Gradients, S-1000 Chromatography, Immunoprecipitation, and Cross-linking—Differential centrifugation and sucrose density gradients were conducted as described previously (37Brown C.R. McCann J.A. Chiang H.-L. J. Cell Biol. 2000; 150: 65-76Crossref PubMed Scopus (62) Google Scholar, 40Brown C.R. Liu J. Hung G. Carter D. Cui D. Chiang H.-L. J. Biol. Chem. 2003; 278: 25688-25699Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). S-1000 chromatography was performed as described (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). Yeast cells expressing Vid24p-HA were grown in YPKG (50 ml) for 2 days and shifted to YPD for 30 min. Cells were harvested and cell lysates were subjected to differential centrifugation. Vid vesicle-enriched pellet fractions were further fractionated on sucrose density gradients. Fractions were collected and proteins from each fraction were blotted with anti-HA, anti-Sec28p, or anti-coatomer antibodies. For immunoprecipitation experiments, Vid vesicle-enriched fractions from the sucrose gradients were pooled, followed by 2% Triton X-100 solubilization. After centrifugation at 13,000 × g for 20 min at 4 °C, the supernatant was then incubated with 2 μl of anti-HA antibodies, followed by 100 μl of a 50% slurry of protein G beads (Amersham Biosciences). Beads were then washed three times and the bound and unbound fractions were detected by Western blotting with anti-HA, anti-Sec28p, anti-FBPase, and anti-coatomer antibodies. Chemical cross-linking experiments were performed on selected fractions as described previously (44Bowser R. Müller H. Govindan B. Novick P. J. Cell Biol. 1992; 118: 1041-1056Crossref PubMed Scopus (88) Google Scholar) with minor modifications. Briefly, DSP was added to 100 μl of the sucrose gradient fraction to a final concentration of 1 mg/ml. Samples were incubated on ice for 45 min and the reaction was quenched via the addition of 50 μl of 0.4 m ammonium acetate. SDS was added to a final concentration of 1%, followed by heating at 65 °C for 10 min. Samples were cooled on ice and 1 ml of immunoprecipitation buffer was added to dilute the SDS. HA antibody was added to the mixture and incubated overnight, and material was captured using protein G-Sepharose. Following washes with IP buffer, proteins were released from the beads via the addition of 1× sample buffer with or without 50 mm dithiothreitol. GFP Studies—GFP microscopy was performed as described (36Huang P.H. Chiang H.-L. J. Cell Biol. 1997; 136: 803-810Crossref PubMed Scopus (80) Google Scholar). FBPase-GFP or Sec28p-GFP were transformed into cells using the PCR-based integration methods described by Longtine et al. (45Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4171) Google Scholar). The PCR was performed using the primers listed in Table 2. Cells were grown under YPKG conditions to induce FBPase. In most studies, cells were shifted to glucose in the presence of the FM 4-64 dye for various periods of time. In some experiments, cells were preincubated with FM 4-64 for 1 h and chased in YPKG without the dye overnight. These cells were then shifted to glucose for various periods of time. Cells were examined using a Zeiss Axiovert s100 (Carl Zeiss Inc., Thornwood, NY) fluorescence microscope and images were taken with a digital camera (Hamamatsu Inc., Japan). Immunofluoresence Microscopy—Δvam3 cells expressing Vid24p-HA were grown in YPKG to induce FBPase. Cells were then shifted to glucose for 30 min and spheroplasted with zymolase. Cells were incubated with anti-HA antibodies at 1:10 dilution or anti-Sec28p antibodies at 1:10 dilution followed by Texas Red-conjugated goat anti-mouse secondary antibodies or fluorescein isothiocyanate-conjugated goat anti-rabbit antibodies at 1:50 dilution. Cells were washed and visualized using fluorescence microscope equipped with a digital camera. SEC28 Is Required for the Vacuolar Pathway of FBPase Degradation—In an attempt to identify molecules that are involved in FBPase degradation and Vid vesicle function, two different approaches were used. The first one utilized genome-wide screening of a deletion library where individual open reading frames were disrupted. Strains that failed to degrade FBPase following a shift to glucose were identified using a colony blot procedure (30Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). One strain that showed a severe FBPase degradation defect was the Δsec28 mutant. SEC28 encodes the ϵ-COP component of the COPI coatomer, a complex that has a known role in vesicular transport from the Golgi to the ER, as well as endocytic trafficking (3Wieland F. Harter C. Curr. Opin. Cell Biol. 1999; 11: 440-446Crossref PubMed Scopus (91) Google Scholar, 4Lee M.C. Miller E.A. Goldberg J. Orci L. Schekman R. Annu. Rev. Cell Dev. Biol. 2004; 20: 87-123Crossref PubMed Scopus (706) Google Scholar, 5Bednarek S.Y. Orci L. Schekman R. Trends Cell Biol. 1996; 6: 468-473Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 7Duden R. Mol. Membr. 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Cell Biol. 1997; 136: 803-810Crossref PubMed Scopus (80) Google Scholar) and proteins from the Vid vesicle fractions were subjected to MALDI analysis. A number of proteins were identified using this approach. These included Sec28p, as well as other subunits of the COPI coatomer complex such as Sec21p and Ret1p. We have shown that FBPase is degraded in the proteasome when glucose is added to cells that are starved for 1 day. In contrast, when 3-day starved cells are shifted to glucose, FBPase is degraded in the vacuole (36Huang P.H. Chiang H.-L. J. Cell Biol. 1997; 136: 803-810Crossref PubMed Scopus (80) Google Scholar). Therefore, to test whether SEC28 functions in the vacuolar pathway or proteasome pathway, the Δsec28 strain was starved of glucose for either 1 or 3 days. Cells were then shifted to fresh glucose and examined for FBPase degradation. For 1-day starved Δsec28 cells, FBPase was degraded at a rate similar to that seen in wild type cells (Fig. 1A). By contrast, when 3-day starved Δsec28 cells were shifted to glucose, FBPase degradation was blocked (Fig. 1A). Therefore, SEC28 is not involved in the proteasome-dependent pathway, but is needed for the vacuolar dependent pathway. SEC28 Plays a Role in the Import of FBPase into Vid Vesicles—For the vacuolar-dependent degradation pathway, FBPase is imported into intermediate carriers know as Vid vesicles (36Huang P.H. Chiang H.-L. J. Cell Biol. 1997; 136: 803-810Crossref PubMed Scopus (80) Google Scholar). If SEC28 is involved in either FBPase import into Vid vesicles or in the formation of Vid vesicles, FBPase should accumulate in the cytos
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