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 cln3ÃÂ cells were fragmented, together they
occupied a large space, which accounted for a significant fraction of the overall cell size
increase in cln3ÃÂ cells. Second, cytosol prepared from cells lacking Cln3p had reduced
vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was
perturbed in cln3ÃÂ 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.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/4795 |
| Date | 25 April 2007 |
| Creators | Han, Bong Kwan |
| Contributors | Polymenis, Michael |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 12245318 bytes, electronic, application/pdf, born digital |
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