Abstract
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 epsilon-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 Deltavph1 mutant. Taken together, our results indicate a strong connection between the Vid trafficking pathway and the endocytic pathway.
Highlights
Characterization of complex I (COPI) vesicles has revealed the presence of a protein complex called “coatomer.” Coatomer is a cytosolic protein complex comprised of seven subunits, ␣-COP (160 kDa), -COP (110 kDa), Ј-COP (102 kDa), ␥-COP (98 kDa), ␦-COP (61 kDa), ⑀-COP (35 kDa), and -COP (20 kDa) [11,12,13]
We propose that Sec28p resides on Vid vesicles, and these vesicles later merge with the endocytic pathway
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
Summary
Strains used in this study Deletion strains derived from BY4742 were from Euroscarf. HLY635 BY4742 ⌬sec HLY1080 HLY1385 HLY 2119 HLY225 ⌬vam HLY1023 HLY1816 RSY1010 RH3517 RH3521 RH3516 YW05 ⌬ise1 ⌬arf HLY228 HLY1386 HYL1817 HLY1816 HLY1422 ⌬ypt7 ⌬ubc1 ⌬vph HLY1412 HLY1463 HLY1793 HLY2113 HLY1747 ⌬pep W303. MAT␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 sec28::kanMX4 MAT␣ leu2⌬0 lys2⌬0 ura3⌬0 FBPase-GFP::HIS3. MAT␣ leu2⌬0 lys2⌬0 ura3⌬0 sec28::kanMX4 FBPase-GFP::HIS3 MAT␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 vam3::kanMX4 FBPase-GFP::HIS3. MATa his3-⌬200 ura leu112 lys801 vid24::TRP1 MAT␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0 vam3::kanMX4. We have determined that FBPase import into Vid vesicles requires the heat shock protein Ssa2p [37], Vid22p [38], and cyclophilin A [39]. 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 ⌬vph 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. 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
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