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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 cln3Ã Â cells were fragmented , together they
occupied a large space , which accounted for a significant fraction of the overall cell size
increase in cln3Ã Â cells . Second , cytosol prepared from cells lacking Cln3p had reduced
vacuolar homotypic fusion activity in cell -free assays . Third , vacuolar segregation was
perturbed in cln3Ã Â 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 . |
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