Han, Bong Kwan (2007-04). The G1 cyclin Cln3p regulates vacuole homeostasis through phosphorylation of a scaffold protein, Bem1p, in Saccharomyces cerevisiae. Doctoral Dissertation.
Thesis
How proliferating cells maintain the copy number and overall size of their organelles is not clear. In the budding yeast Saccharomyces cerevisiae the G1 cyclins Cln1,2,3p control initiation of cell division by regulating the activity of the cyclin-dependent kinase (Cdk) Cdc28p. We show that Cln3p controls vacuolar (lysosomal) biogenesis and segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar fragmentation. Although the vacuoles of cln3A?A? cells were fragmented, together they occupied a large space, which accounted for a significant fraction of the overall cell size increase in cln3A?A? cells. Second, cytosol prepared from cells lacking Cln3p had reduced vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was perturbed in cln3A?A? cells. Our findings reveal a novel role for a eukaryotic G1 cyclin in cytoplasmic organelle biogenesis and segregation. Furthermore we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p/Cdc28p complex. The Cdc42p GTPase is known to be required for vacuole fusion. Our results suggest that Ser72 on Bem1p is phosphorylated by Cdc28p in a Cln3p-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdkconsensus site located in the first SH3 domain of Bem1p, suppressed vacuolar fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence of a non-phosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a mechanism that links cyclin-dependent kinase activity with vacuole fusion through Bem1p and the Cdc42p GTPase cycle.
