Roles Of Rab GTPase Vps21 And Its Upstream And Downstream Proteins In Autophagy In Saccharomyces Cerevisiae

Autophagy is a conserved intracellular degradation pathway, which helps cell to maintain homeostasis by degrading proteins and damaged organels and recycling of nutrients. In higher eukaryotes, autophagy is involved in multiple physiological and pathological processes, such as development, immunity, programmed cell death, cacer and neurodegeneration and so on.In the autophagy process, phagaphores expand and engulf parts of cytoplasm to form double memrane autophagosomes, which fuse with the vacuole where they are degraded. This process is regulated by multiple autophagy-related proteins. As it is also a membrane trafficking and fusion event, many Rab GTPases and SNAREs, which are regulators of vesicle trafficking, are reported to be involved in autophagy. For example, in budding yeast, Rab1/Ypt1, with different modules, regulates ER to Golgi vesicle transport and the initial steps of autophagy and Rab7/Ypt7, with the same module, regulates the fusion of late endosomes and autopahgosomes with the vacuole. This study investigates the possible roles of Rab5 and its up-and downstream proteins, which function before Ypt7 in endocytosis in Saccharomyces cerevisiae in endocytosis, in autophagy and its mechanism. The following main results are obtained:1. Absence of Vps21 results in accumulation of clusters of autophagosomes outside the vacuoleThere are three Rab5 homologues in budding yeast:Vps21, Ypt52 and Ypt53, and Vps21 is the most impotant one for endocytosis. The effects of loss of Rab5 on autophagy are tested and results show that the absence of Vps21 but not Ypt52 or Ypt53 results in decreased alkaline phosphatase activity, defects in Apel maturation and GFP-Atg8 degradation, but simutaneously absence of Ypt52 and Vps21 exacerbates the defects. Using fluorescencent microscopy, GFP-Atg8 is observed to accumulate on vacuole membrane as crescent-like structures in cells without Vps21. These structures are clusters of autophagosomes as shown by transmissional electron microscopy (TEM). These results suggest that the absence of Vps21, but not Ypt52 or Ypt53, results in clusters of autophagosomes accumulated outside the vacuole.2. Absence of Vps9 results in similar defects as the absence of Vps21 and the defects can be suppressed by overespression of Vps21In order to fulfill their tasks, Rab proteins need to switch from the GDP-bound "inactive" form to the GTP-bound "active" form, which is regulated by guanine nucleotide excahge factors (GEFs). Results here show that absence of Vps9, the Vps21 GEF in endocytosis, results in similar autophagy defects as the absence of Vps21:decreased alkaline phosphatase activity and accumulation of autophagosome clusters. And the defects in vps9Δ cells can be suppressed by overexpression of Vps21. The absence of Mukl, the newly indentified Rab5 GEF in budding yeast, does not show any defects, but simutaneously deletion of MUK1 and VPS9 results in more severe autophagy defects than VPS9 single deletion mutants. And the defects in the double mutants can not be rescued by overexpression of Vps21. Thess suggest that Rab-GEF relationship of Vps9 and Vps21 still exists in autophagy pathway and Mukl may have other roles, for example, stablizing active Vps21 or activiting other Rabs.3. Loss of Vps21 downstream factors result in similar autophagy defects as the absence of Vps21Rab protein Vps21, GEF Vps9 and effectors make up the functional module of Vps21. Accordingly, Rab protein Ypt7, GEF Monl-Cczl and effectors make up the functional module of Ypt7, regulating the fusion of late endosomes and atutophagosomes with the vacuole. In endocytosis, factors that function downstream of Vps21 are:tether CORVET complex, adaptor protein Vac1, t-SNARE Pep 12 and SM family protein Vps45. Results show that the absence of Vps3 and Vps8 (CORVET specific subunits), Vacl, Pep12 and Vps45 result in similar defects as the absence of Vps21:defects in Apel maturation and GFP-Atg8 degradation and GFP-Atg8 accumulation on the vacuole membrane, and the absence of Vps3, Vacl or Pep12 alone shows the most severe defects. The defects in Vps21 module protein mutants are different from those in Ypt7 module mutants, which show dispersed GFP-Atg8 puncta in cytosol. The above results suggest that not noly Vps21 and Vps9, but the Vps21 endocytic module regulates autophagy.4. Localization of Vps21 to PAS is increased in Vps21 downstream effector mutantVps21 localizes on endosomes to regulate endocytosis and it may localize to phagophore assembly site (PAS), where autophagosomes are generated, to regulate autophagy. The localization of Vps21 at PAS can be an envidence for a direct role of Vps21 in autophagy. Results show that Vps21 partially localizes to PAS at a low percentage in wild type. The low percentage of colocalization may be due to the fast formation of autophagosomes and transiently localization of Vps21 on the PAS. In consistent with this hypothesis, the colocalization between Vps21 and PAS markers increased dramaticly in PEP12 deletion mutant, in which both Vps21 and autophagosomes are accumulated. This suggests that Vps21 localizes to PAS and probably regulates autophagy in this way.5. Vps21 module may regulate autophagy through PI3K complexThe mechanism for the function of Vps21 module in autophagy is investigated by yeast two hybrid screening of interacting proteins. Results show that:most of Vps21 module proteins interact with one or several of Atg17, Atg20 and Atg24, in which Vps8 shows the strongest interaction with all three proteins. This indicates that Vps21 module may be recuited onto phagophores via interaction with Atgl7 scanfold system (Atgl7, Atg20 and Atg24).Vps34 is the catalytic subunit of PI3K complex, which is important for both endocytosis and autophagy. Yeast two hybrid screening results show that Vps8 interacts with Vps34. Physical interactions between Vps21 module proteins and PI3K subunits are then investigated and results show that Vps8 interacts with three of the PI3K complex:Vps34, Vps15 and Vps30 (strongest interaction with Vps15); Vps21 also interacts with Vps15. And the interaction between Vps8 and Vps34 is dependent on Vps21 and Pep 12-but not Ypt7 and its downstearm t-SNARE Vam3, indicating that Vps21 module interacts with PI3K complex in toto. Vps21 module also regulates the localization of Vps34, and loss of Vps21 module proteins results in mislocalization of Vps34 from PAS. Further studys show that the effect of Vps8 domain deletion mutants on Vps8-Vps34 interaction, Vps34 localization and autophagy is different. In summary, Vps8 N-terminal domains are important for interactions of Vps8 with Vps34 and Vps21, but the C-terminal domains are required for Vps34 localization and autophagy. These results suggest that Vps21 module may involve in autophagy through regulation of Vps34 localization.6. Vps21 module regulates the sealing of autophagosomesThe protein protection assay is used to distinguish whether the autophagosomes are closed. Using this method, we show that Vps21 module functions in autophagosome formation with unsealed autophagosomes and before Ypt7 module as it does in endocytic pathway. Combinding with the accumulation of clustered autophagosomes in Vps21 module mutants as shown by TEM, we conclude that Vps21 module functions in the final sealing step of auutophagosome formation.Taken together, we show that Vps21 endocytic module also regulates autophagy. We then show that Vps21 module may involve in autophagy through interaction with PI3K complex. Further study shows that Vps21 module is involved in the final sealing step of autophagosome formation and functions before Ypt7 module in autophagy pathway. This study paved the role for further investigation of how Vps21 module functions in autophagy and provides useful references for studying the mechanism of other Rabs in autophagy.