Role of trans-Golgi Network and Endosomal Adaptors in Growth of Saccharomyces cerevisiae

Abstract:

In the yeast Saccharomyces cerevisiae, trafficking of a variety of proteins between the cell surface, trans-Golgi network (TGN), endosomes, and vacuole is regulated by clathrin adaptors. In this study, we focused on the role of the TGN-endosomal clathrin adaptors Gga1, Gga2, AP-1, Ent3, and Ent5 on the nitrogen-regulated growth of S. cerevisiae. Evidence suggests that the trafficking of the major amino acid and nitrogen permeases used for cell growth are regulated by these clathrin adaptors. We hypothesized that distinct combinations of adaptors may regulate nitrogen permease trafficking under different nitrogen conditions, leading to distinct effects on cell growth. To discern the role of clathrin adaptors in nitrogen-regulated growth, we have constructed various single, double, and triple clathrin adaptor deletion mutants and tested their effects on growth in preferred and non-preferred nitrogen sources. Growth assays were performed in 96 well plates and growth curves were plotted to visualize the differences in growth rate in mutant vs. wild-type cells. Our results show that the single deletion of either ent3, ent5, or apl2 (AP-1 subunit) did not impact cell growth in preferred or non-preferred nitrogen sources. Double deletions of ent3 and ent5 but not apl2 with ent3 or ent5 had reduced growth in cells in both preferred and nonpreferred nitrogen sources. A triple deletion of ent3, ent5, and apl2 had the greatest growth defect in both preferred and nonpreferred nitrogen sources. These results suggest that Ent3 and Ent5 are functionally redundant and complementary to AP-1 function, and that cell growth requires AP-1 and Ent3 or Ent5.  Moreover, these clathrin adaptors’ role in cell growth is not linked to their function in regulating specific nitrogen permeases and suggests a more general function in cellular processes that may affect metabolism and/or cell cycle regulation.