Roles Of Specific Subunits Of Trapp Complex In Vesicle Trafficking And Autophagy In Saccharomyces Cerevisiae

Vesicle trafficking and autophagy are basic physiological processes in eukaryotes for cell survival. Vesicle trafficking is an important way for the transport of substances within the cell, while autophagy is an essential cellular degradation process that eliminates obsoletes or damaged cytoplasmic materials to maintain intracellular homeostasis.Vesicle transport among different organelles in yeast is regulated by a molecular switch named Ypt/Rab GTPases, which are activated by Guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs) to cycle between GTP-bound form and GDP-bound form. TRAPP (Transport protein particle) complex is one of the large multisubunit complexes and is implicated in tethering vesicles. Three TRAPP complexes (Ⅰ, Ⅱ and Ⅲ) are reported in yeast for their GEF activities for GTPases Yptl and Ypt31/32. TRAPPI regulates endoplasmic reticulum(ER)-to-Golgi trafficking by activating Ypt1, TRAPPII regulates Golgi-to-plasma membrane and endosome-to-Golgi trafficking by activating Ypt31/32. TRAPPIII regulates autophagy by activating Yptl Trs85is a specific subunit of TRAPPIII complex which has important roles in autophagy. There are also data showing that the overexpression of Trs85can suppress the growth defect of bet3ts and bet5ts (Bet3and Bet5is the subunit of TRAPP complex) mutants at high temperature. Others further reported that the GFP-Sncl (Sncl is a v-SNARE protein) transport was blocked in trs85A mutant and the phenotype is similar to the mutant of TRAPPII-specific subunits. These genetic evidences indicate that Trs85participates in vesicle trafficking with unknown mechanism. Trs130is a specific subunit of TRAPPII complex, and some data suggest that Trs130is involved in the transport of substance to exit the Golgi. It is unknown whether Trs130and other TRAPPⅡ-specific subunits regulate autophagy. Recently, Ypt31/32was shown to regulate autophagy. It was speculated that TRAPPII complex may regulate autophagy through its substrate Ypt31/32. In order to globally elucidate the functions of TRAPPIII and TRAPPII (both with specific TRAPP subunits) in both vesicle trafficking and autophagy, this study focuses on the functions and mechanisms of Trs85in vesicle trafficking and Trsl30in autophagy. The main results are listed below:1. Trs85played a role in ER-to-Golgi vesicle trafficking in yeastGFP-Snc1was used as a probe to reflect the process of vesicle trafficking in yeast. Results found are:(1) GFP-Sncl tagged strains became temperature-sensitive when TRS85was deleted, the resulting strain designated as trs85Δts;(2) GFP-Sncl accumulated in the ER and did not recycle to the PM in trs85Δts mutant cells after incubated at high temperature treatment. Similar GFP-Sncl phenotype was found in yptlts mutant at the restrictive temperature. This internal GFP-Sncl phenotypes in both mutants were different from those in ypt31A/32ts and trs130ts mutant cells, in which GFP-Sncl accumulated in endosomes with defect to reach the PM at the restrictive temperature. Results suggest that Trs85and Yptl regulate ER-to-Golgi transport; whereas Trs130and Ypt31/32regulate endosome-to-Golgi transport.2. Grouping Yptl with Trs85and Ypt31/32with Trs130in vesicle traffickingThere are similar GFP-Sncl phenotypes between trs85Δts and yptlts mutant. In order to know whether Trs85functions through Yptl in vesicular transport, Yptl and Ypt31/32were overexpressed in trs85Δts and trs130ts mutant cells. Results are:Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs85Δts mutant cells at the restrictive temperature, while Ypt31/32did not. In contrast, Ypt31/32, but not Yptl restored the intracellular trafficking of GFP-Sncl and the growth of trs130ts mutant cells at the restrictive temperature. These results suggest that Trs85or TRAPPIII regulates ER-to-Golgi transport through Yptl while Trs130regulates endosome-to-Golgi transport through Ypt31/32.3. Trs130regulated the recruitment of Atg8and Atg9from the trans-Golgi to the PAS in autophagy.The Pho8A60assay is a quantitative method to detect autophagy. When autophagy was induced at a non-permissive temperature (NPT), the Pho8Δ60activity in trs130ts mutant is much lower than that in wild-type. Immunoblot assay was further applied to determine GFP-Atg8degradation and prApe1maturation in the trs130ts mutant in the process of autophagy. Results showed that prApel maturation and GFP-Atg8degradation were impaired in trs130ts cells under both rich and starvation conditions at NPT. By fluorescence microscopy, GFP-Atg8was found to accumulate in the cytosol as multiple dots at NPT. The Atg9is the only transmembrane protein among Atg proteins and Atg8transport depends on Atg9. Using TAKA assay, we found that Atg9anterograde transport was defective in trs130ts mutant at NPT independent of culture medium. These results indicate that autophagy processes are blocked in trs130ts mutants.Atg-related protein Atg11and Atg17are scaffolding proteins, and both can recruit other Atg proteins to the PAS in the Cvt pathway and autophagy. GFP-Atg8in trs130ts mutant still accumulated as multiple dots in the absence of ATG11and ATG17. This indicates Trsl30functions upstream of the recruitment of Atg proteins to the PAS. Sec7is indispensable for secretion and localizes to the trans-Golgi. Using Sec7-DeRed as a trans-Golgi marker, Atg8and Atg9were found to be partially trapped in the trans-Golgi, not like the few localization of Atg8and Atg9in the trans-Golgi in wild-type. In summary, autophagy was impaired in trs130ts mutant and Trs130regulates the recruitment of Atg8and Atg9from the trans-Golgi to the PAS.4. Trs130mediates autophagy through the activity of Yptt31/32Untill now, both Trs130and Ypt31/32regulate autophagy. Trs130mediates vesicle trafficking through Ypt31/32, but it is unclear whether Trs130mediates autophagy through Ypt31/32. In this study, we found that the overexpression of Ypt32, Ypt31or the GTP-bound form of Ypt31, but not Yptl or the GTP-bound form of Ypt31, rescued autophagy defects in trs130ts mutant cells. We conclude that Trs130participates in autophagy through the activity of Ypt31/32and suggest that the GEF-GTPases relationship existing in both vesicle trafficking and autophagy.This study elucidate the functions and mechanisms of Trs85and Trs130in vesicle trafficking and autophagy respectively. The results obtained here provide comprehensive experimental evidence for the roles and mechanisms of TRAPPIII and TRAPPⅡ in vesicle trafficking and autophagy, which will facilate the studies of the functional relationships among TRAPP complexes.