Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth.
The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.
Richman
Tamara J
TJ
Department of Microbiology and Molecular Genetics and the Markey Center for Molecular Genetics, University of Vermont, 95 Carrigan Dr., 202 Stafford Hall, Burlington, VT 05405, USA.
Toenjes
Kurt A
KA
Morales
Sergio E
SE
Cole
Karen C
KC
Wasserman
Ben T
BT
Taylor
Chad M
CM
Koster
Jacob A
JA
Whelihan
Matthew F
MF
Johnson
Douglas I
DI
D002453:Cell Cycle / Q000235:genetics / Q000502:physiology*; D049452:Green Fluorescent Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D009711:Nucleotides; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology*
C105191:Bni1 protein, S cerevisiae; D020730:Guanine Nucleotide Dissociation Inhibitors; D008840:Microfilament Proteins; D009711:Nucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae