Bem1p, a scaffold signaling protein, mediates cyclin-dependent control of vacuolar homeostasis in Saccharomyces cerevisiae.
How proliferating cells maintain the copy number and overall size of their organelles is not clear. We had previously reported that in the budding yeast Saccharomyces cerevisiae the G1 cyclin Cln3p is required for vacuolar (lysosomal) homotypic fusion and loss of Cln3p leads to vacuolar fragmentation. The Cdc42p GTPase is also required for vacuole fusion. Here we show that the scaffold protein Bem1p, a critical regulator of Cdc42p activity, is a downstream effector of Cln3p and the cyclin-dependent kinase (Cdk) Cdc28p. Our results suggest that Bem1p is phosphorylated in a Cdk-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdk-consensus 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 nonphosphorylatable 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.
Han
Bong-Kwan
BK
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA.
Bogomolnaya
Lydia M
LM
Totten
James M
JM
Blank
Heidi M
HM
Dangott
Lawrence J
LJ
Polymenis
Michael
M
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D017489:CDC28 Protein Kinase, S cerevisiae; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D006706:Homeostasis / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism*; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C079895:CLN3 protein, S cerevisiae; D016213:Cyclins; D029701:Saccharomyces cerevisiae Proteins; C073129:BEM1 protein, S cerevisiae; D017489:CDC28 Protein Kinase, S cerevisiae; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae