Roles Of Essential Subunits Of Trapp Complex, Bet5 And Trs20, In Autophagy In Saccharamyceres Cerevisia

In Saccharomyces cerevisiae, Ypt/Rab small GTPases regulate vesicle trafficking between different organelles. Small GTPases recycle between GTP-bound status (active form) and GDP-bound status (inactive form). This recycling is controlled by guanine nucleotide exchange factors (GEFs) and GTPase active proteins (GAPs). In yeasts, Transport Protein Particle (TRAPP) is a multiple-subunit complex which takes part in vesicle trafficking and autophagy as GEFs of small GTPases Ypt1 and Ypt31/32. Vesicle trafficking is an important pathway of intracellular transport, which maintains the normal life of cells, while autophagy is a major homeostatic mechanism that favors adaption and survival under stress conditions.According to the latest report, there are three kinds of TRAPP complexes. TRAPPⅠ is constituted of five essential subunits, Trs20, Trs31, Trs23, Bet3, Bet5; TRAPP Ⅱ is constituted of TRAPPⅠ and other five specific subunits, Trs120, Trs130, Trs65, Trs33, Tca17; and TRAPPⅢ is constituted of TRAPPⅠ and one specific subunit Trs85. In yeasts, TRAPPI functions as a GEF of Ypt1, regulating vesicle trafficking from endoplasmic reticulum (ER) to Golgi; TRAPPⅡ functions as a GEF of Ypt31/32, regulating vesicle transport from Golgi to plasma membrane and endosome to Golgi traffic, and also taking part in autophagy; TRAPPⅢ regulates both vesicle trafficking and autophagy by activating Ypt1. Results of TRAPPⅡ and TRAPPⅢ in vesicle trafficking and autophagy came from studies on the their specific subunits. However, roles of the five essential subunits (also composited of TRAPPⅠ) in autophagy are not reported. Preliminary study in this lab found that the mutation of Bet3, an essential subunit of TRAPP complex, imapired autophagy. Furthermore, overexpression of Ypt1, but not Ypt31/32, suppressed the autophagy defect in bet3ts. It is urgent to study the roles of other four essential common subunits of TRAPP complex in autophagy.BET5 and TRS20 are two essential genes for yeasts, and the absence of either one results in lethality. Temperature sensitive mutants of the two genes are available for further study. This study focused on the autophagy defect in bet5ts and trs20ts mutants and the suppression of Ypt1 and Ypt31/32 on their autophagy defect. The obtained results will briefly implicate how Bet5 and Trs20 function in autophagy. Results showed that autophagical processes were impaired in bet5ts and trs20ts mutants:in nutrient rich conditions, the selective autophagy marker Ape1 did not mature in these two mutants; under starvation conditions, the nonselective autophagy marker GFP-Atg8 accumulated in the cytoplasm and was not degraded in the vacuole at 26℃. In addition, the anterograde transport of GFP-Atg9 to the pre-autophagosomal structure/phagophore assembly site (PAS) is reduced in these two mutants. Furthermore, overexpression of Yptl suppressed temperature sensitivity of bet5ts and trs20ts mutants growing at restrictive temperature in rich medium. At the same time, overexpression of Yptl also restore the transport of GFP-Atg8 to the vacuole for degradation in these two mutants. All these results indicate that vesicle trafficking and autophagy are close related, and Bet5 and Trs20 may participate in autophagy through Ypt1.