Studies Of Trapp Complex Subunits In Vesicle Trafficking And Autophagy VIA RAB Gtpases In Saccharomyces Cerevisiae

Vesicle trafficking and autophagy are vital physiological processes in eukaryotic cells. Vesicles bud off from one membrane compartment and fuse with another, carrying membrane components and soluble molecules referred to as cargo. Whereas autophagy is a cellular process in which cytoplasmic contents are degraded within the lysosome/vacuole, and the resulting building blocks are recycled.In yeast, Rab (named Ypt in yeast) GTPases regulate vesicle trafficking. Rab/Ypt is activated from the inactive GDP state to the active GTP state by guanine nucleotide exchange factor (GEF). Three transport protein particle (TRAPP) complexes (?, ? and ?) are reported for their GEF activities for Yptl and Ypt31/32. TRAPP I is required for endoplasmic reticulum-to-Golgi transport by activating Yptl. TRAPP ?is required for Golgi-to-plasma membrane and endosome-to-Golgi trafficking, and autophagy by activating Ypt31/32. TRAPP ? functions in autophagy through Yptl, and endoplasmic reticulum-to-Golgi transport. However, the composition and functions of TRAPP complexes are controversial. Recently, Tea17 was also reported to be a TRAPP ?-specific subunit. Trs33, originally reported as a TRAPP ? subunit, is suggested to be a TRAPP ?-specific subunit based on genetics study. Tea17 and Trs33 are both required for TRAPP? assembly. TCA17 or TRS33 deletion results in defects of vesicle trafficking and autophagy. However, the precise mechanism has not been thoroughly studied, except that Ypt31 but not Yptl suppresses the temperature sensitivity of mutant growth. In addition, the data of our lab showed that the common subunits of TRAPP complexes (Bet3, Bet5 and Trs20) also participate in autophagy. In this study, we focus on the role of Tea17 and Trs33 in vesicle trafficking and autophagy, and the role of Trs23 and Trs31 (another two common subunits of TRAPP complexes) in autophagy.The obtained results were listed below:1. Tea 17 and Trs33 participate in vesicle trafficking through Ypt31Wild-type and TCA17 or TRS33 deletion mutants were tagged with green fluorescence protein (GFP) for vesicle trafficking marker protein Snc1. The defects of growth phenotype and GFP-Snc1 transport were examined in these strains. Furthermore, the suppression effect of different Rab GTPases on growth and vesicle trafficking defects in these deletion mutants were investigated. Results showed that overexpression of Ypt31, but not Ypt1, suppresses the temperature sensitivity of growth and the defect of GFP-Snc1 transport in TCA17 or TRS33 deletion mutants. These results suggest that Tea17 and Trs33 participate in vesicle trafficking through the activity of Ypt31.2. Tca17 and Trs33 mediate autophagy through Ypt31Wild-type and TCA17 or TRS33 deletion mutants were tagged with green fluorescence protein (GFP) and red fluorescence protein (RFP) for autophagy marker protein Atg8 and Ape1, respectively. The defects of growth phenotype and autophagy in nutrient-rich and starvation conditions were examined in these strains. Deletion of TCA17 results in the defect of Ape1 maturation in the Cvt pathway. TCA17 or TRS33 deletion also results in the temperature sensitivity of growth and the defect of transportation and degradation of GFP-Atg8. Overexpression of Ypt31, but not Ypt1, suppresses the temperature sensitivity of growth and the defect of transportation and degradation of GFP-Atg8 in TCA17 or TRS33 deletion mutants. These results indicate that Tea17 and Trs33 mediate autophagy through the activity of Ypt31.3. Trs23 and Trs31 regulate autophagy through Ypt1TRAPP? and TRAPP? specific subunits have been reported to be involved in autophagy. Studies on how TRAPP common subunits participate in autophagy become necessary. Mutation in TRS23 or TRS31 results in the temperature sensitivity of growth. Strains were tagged with autophagic markers through mating and dissection. These mutant cells are defective in transportation and degradation of GFP-Atg8 and maturation of Ape1. Ypt1, but not Ypt31, suppresses the temperature sensitivity of growth, the defect of transportation and degradation of GFP-Atg8 and the maturation of Ape1 in TRS23 or TRS31 mutants. We propose that Trs23 and Trs31 regulate autopagy through Ypt1.These results are important for interpreting the mechanisms of different TRAPP subunits in vesicle trafficking and autophagy in Saccharomyces cerevisiae. The dual roles of different TRAPP complex subunits in both vesicle trafficking and autophagy also provide further evidences to support the emerging roles of Ypt/Rab GTPases and their activators in autophagy. However, the linkage between vesicle trafficking and autophagy still needs further investgation.