The Complementation Of The Small Gtpases YPT1for YPT6in Exocytosis And Autophagy In Saccharomyces Cerevisiae

Ypt6and Yptl are two small GTPases, which are two members of the Rab family in Saccharomyces cerevisiae. Like other small GTPases, they also recycle between GTP-binding state and GDP-binding state. In GTP-binding state, Ypt6and Yptl interact with their downstream effectors to establish functional activities. Rgp1and Ric1are two subunits of the Ypt6GEF (Guanine exchange factor) complex, which converts Ypt6from GDP-binding form to GTP-binding form. Ypt6is not necessary for living in Saccharomyces cerevisiae. Like Ypt6mutation, the absence of Rgpl or Ricl also results in temperature sensitivity, which can be rescued by the overexpression of Ypt6. Physiological and biochemical data suggest that Ypt6is involved in the early secretory pathway:from ER to Golgi, or from cis-Golgi to medial-Golgi, or both. In YPT6, RGP1or RIC1deletion mutants, the secretion and maturation of several peptidases such as invertase and CPY (Carboxypeptidase Y) are blocked. The vacuolar protein CPY accumulates to form exceptive ER or cis-Golgi, and the deletion of YPT6may lead to the CPY’s location to the cell surface by default. Additionally, it has been assumed that Ypt6may relate to autophagy, and the absence of Ypt6, Rgpl or Ricl will affect the selective-Cvt (Cytoplasm to vacuole targeting) pathway, but has no effect on the nonselective-autophagy.It has been reported that the overexpression of Yptl in ypt6ts strain can suppress the growth defect under high temperature, while the mechanism for it remains unclear. Yptl was the first Rab GTPase to be isolated and characterized in yeast. It locates in ER and Golgi, and plays a role in the transportation from ER to Golgi. It also affects the formation and function of microtubules directly or indirectly, regulates the fusion of the membrane. The lack of growth in ypt6ts may be related to many failures of physiological processes. Yptl may play a role in suppressing these defects.In ypt6ts related mutants such as rgpl△and ricl△, Yptl may also play the same role.This study mainly focuses on the functional defects in exocytotic and autophagy caused by the mutation or deletion of Ypt6and the related Rgp1/Ricl, and the complementation of Ypt1to these defects. The results suggest that the absence of Ypt6and Rgpl/Ricl may block the transportation of exocytotic proteins, such as Sncl, to induce the growth sensitivity. However, overexpressing Ypt1in these mutant cells suppresses transportation defects and growth sensitivity. The FM4-64staining indicated that overexpression of Yptl can also recover the morphology of vacuole in the mutants. The absence of Ypt6or Ricl was reported to affect the Cvt pathway in vegetative phase but not the autophagy under nutrient starvation. This study found the autophagy in ypt6ts and the related mutants mainly was also impaired with retarded degradation GFP-Atg8in shorter starvation treatment. The overexpression of Yptl in these mutants also suppresses the growth and autophagy defect at restrictive temperature. Apel is another protein that can be used to trace the selective-Cvt pathway and the nonselective-autophagy. In this study, whether in rich or starvation conditions, the maturation of Ape1was affected in ypt6ts and the related mutants, and the overexpression of Yptl can suppress the defect. In addition to these, this study also found that overexpression of Yptl can completely suppress the growth of ypt6ts and the related mutansts in high temperature condition, but it cannot restore the proper localization of Ypt6in these mutants, which indicates that Yptl functions independent of Ypt6, but with some redundancy in the function of Ypt6.In conclusion, Yptl can play a role in suppressing the defects of ypt6ts and the related mutants in exocytosis and autophagy, which are supposed to be intimately related. In Saccharomyces cerevisiae, the overexprssion of Yptl can restore growth but not the localization of Ypt6in Ypt6and related mutants, indicating Yptl has some reductant functions of Ypt6to maintain the normal physiological processes. These results partially explore the the mechanism of Yptl in recovering the temperature sensitivity in ypt6ts and related mutants of its GEF.