Studies On Functions Of Three Subunits Of Trapp Complexes In Vesicle Trafficking And Autophagy In Saccharomyces Cerevisiae

Vesicle traffic and autophagy are membrane trafficking pathways vital for cell survival. Vesicle traffick mainly carries out the cargo proteins from one compartment to another, while autophagy is a catabolic process that degades cargoes ranging from organelles to protein aggregates or bacteria, which is important for maintaining cellular homeostasis.Membrane trafficking in budding yeast is regulated by Ypt/Rab GTPases, which are activated by Guanine nucleotide exchange factors (GEFs) and inactivated by GTP hydrolysis through GTPase-activating proteins (GAPs). TRAPP (Transport protein particle) complexes function as multimeric tethering factors, at the same time, they are GEFs for Ypt/Rab GTPases in yeast. Currently there are three TRAPP complexes (TRAPPI, TRAPPⅡ and TRAPPⅢ) reported in yeast, which act in ER-Golgi trafficking, Golgi trafficking and autopahgy respectively. All three complexes share six subunits:Bet3, Trs23, Bet5, Trs31, Trs20and Trs33to form TRAPPI core complex, with the front four subunits being essential for Yptl GEF activity in vivo. Trs65(not present in higher eukaryotes), Trs120, Trs130and Tca17are specific to TRAPPⅡ, whereas Trs85is specific to TRAPPⅢ. Bet3as a common essential TRAPP subunit, plays vital roles in vesicle trafficking. Trs65is required for TRAPPⅡ assembly, whereas Trs85mainly plays roles in autophagy. There are more and more evidences to show that Rab/Ypt GTPases and their regulator GEFs are involved in autophagy, in additional to their roles in vesicle trafficking. Till now, there is no study on comparing the roles of a TRAPP subunits in both vesicle trafficking and autophagy. This study was going to examine and compare the roles of three representative subunits, Bet3, Trs65and Trs85in vesicle trafficking and autophagy. The obtained results were listed below:1. Trs65, Trs85and Bet3are involved in intracellular vesicle traffickingUnder vegetative conditions, TRS65deletion doesn’t impair vesicle trafficking but TRS85deletion does with accumulation of GFP-Sncl inside cells. TRS65and TRS85double deletion lead to more severe vesicle trafficking phenotypes. Part of the accumulated GFP-Sncl in trs85Ats and trs65Ats trs85Ats strains was proved to be on the ER. In contrast, under nitrogen starvation conditions, TRS65deletion impairs vesicle trafficking with blockage in protein transporting to vacuole. There are reports for Bet3’s role in vesicle trafficking. When CPY-GFP was integrated into bet3ts strain, CPY was accumulated in bet3ts strain cells under both rich and starvation conditions. This phenotype in bet3ts strain is like that in yptlts strains.2. TRAPP-specific subunits and their corresponding Ypt GTPases regulate vesicle trafficking in groupYptl can suppress the defects of growth, vacuole morphology and vesicle trafficking in mutation of TRAPPⅢ-specific subunit Trs85, while Ypt31can suppress those defects in mutation of TRAPPⅡ-specific subunit Trs130, but not the opposite.3. Trs65and Trs85are involved in the transport of non-Atg protein to the vacuole under starvation conditionsCells integrated with GFP-SNC1or GFP-SNC1-PEM were shifted to SD-N liquid media to induce autophagy. Result showed that the trs85Ats and trs65Ats trs85Ats strains respond to starvation treatment more slowly than trs65Ats and their wild type strains do. Furthermore, when the cells integrated with GFP-Sncl-PEM under nitrogen starvation conditions for20hours were stained with FM4-64, the vacuoles of trs85Ats, and trs65Atrs85Ats strains are stained only on the vacuolar membrane, while the vacuoles of trs65Ats and their wild type strain are filled with red signals. Although TRS65deletion doesn’t impair autophagy significantly, our results show that GFP-Sncl accumulated in trs65A strain after nitrogen starvation treatment. These results indicate Trs65and Trs85play a key role in the transport of non-Atg protein to the vacuole under starvation conditions.4. Trs85mutation or Trs65and Trs85double mutation affect the vacuole morphology under nitrogen starvation conditionsThe single round vacuole appears in each cell when wild type cells are treated under nitrogen starvation conditions, but Trs85mutation or Trs65and Trs85double mutation delayed the forming process of single round vacuoles, There are significant differences on single round vacuole ratio between wild type and mutants. This indicates that Trs85or Trs65and Trs85together play crucial roles in single round vacuole forming in cells. 5. Bet3is involved in the Cvt pathway and macroautophagy partially through YptlGFP-Atg8, Atg9-3×GFP and RFP-Apel were integrated into bet3ts and its wild type strain. We found in bet3ts strain under both rich and starvation conditions, prApel maturation and GFP-Atg8degradation impaired. the ratio of the colocalization between Atg8and Apel decreased drastically, and the size and brightness of Atg9puncta decreased. This indicates Bet3is involved in the Cvt pathway and macroautophagy. Bet3is a common subunit for TRAPP complexes, which function as GEFs for Yptl and Ypt31/32. It is possible that Bet3affect autophagy through Ypt GTPases. To verify this possibility, Yptl and Ypt31were overexpressed in bet3ts strain. Under vegetative conditions, Yptl or Ypt31overexpression can’t suppress the autophagy defects in bet3ts strain. Yptl and Ypt31co-overexpression simultaneously, or overexpression of GTP-form of Yptl or Ypt31doesn’t help; Under nitrogen starvation conditions, Yptl not Ypt31can partially suppress the autophagy defects in bet3ts strain. When Ypt31was overexpressed together with Yptl, it could enhance the suppression effect of Yptl.The roles of different TRAPP complexes subunits playing in vesicle trafficking and autophagy studied here will help to build the linkage between vesicle trafficking and autophagy.