Bem1p, a scaffold signaling protein, mediates cyclin-dependent control of vacuolar homeostasis in Saccharomyces cerevisiae

Abstract

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.

Yeast vacuole fusion: A model system for eukaryotic endomembrane dynamics

“…Mapping interaction partners during the fusion reaction should provide some insight into this question. It should be mentioned that Cdc42 has also been encountered in a different context; Cdc42 overexpression rescues the fragmentation phenotype observed in cln3D cells, 140 suggesting a connection between cell cycle control and actin dynamics. Furthermore, S. pombe Cdc42 is connected genetically to Vtc1/Nrf1, 164 a factor implicated in the fusion process (see below).…”

“…Subsequently, a protein required for cell polarization during budding and mating, Bem1, was identified as a potential target of Cln3-mediated phosphorylation. 140 Vacuoles lacking Bem1 are fragmented and deficient in fusion. Whereas one report identifies S72 as a potential phosphorylation site in Bem1, 140 another could not confirm that this residue is critical for Bem1 function.…”

“…140 Vacuoles lacking Bem1 are fragmented and deficient in fusion. Whereas one report identifies S72 as a potential phosphorylation site in Bem1, 140 another could not confirm that this residue is critical for Bem1 function. 141 Until now, the molecular function of Bem1 or additional Cln3-cyclin-dependent kinase targets have not been clarified.…”

Morphogenesis and the Cell Cycle

“…For example, CDK could promote assembly of the PAK-Bem1p-GEF complex to enable the positive feedback loop illustrated in Figure 4. Like Cdc24p, Bem1p and the PAKs Ste20p and Cla4p are CDK substrates, but mutation of putative or mapped phosphorylation sites has thus far failed to reveal any role for those phosphorylations in polarization (Oda et al 1999;Ubersax et al 2003;Han et al 2005).…”

Ribosome Biogenesis Is Sensed at the Start Cell Cycle Checkpoint

“…Vacuole enlargement can have a major impact on cell volume, and indeed, at least some of the increased cell size in cln3 null cells (Cross, 1988;Nash et al, 1988) is due to vacuolar enlargement (Han et al, 2003(Han et al, , 2005. Vacuoles are analogous to mammalian lysosomes and function as a repository for metabolites and low-molecularweight compounds.…”