A mechanism of Bud1p GTPase action suggested by mutational analysis and immunolocalization.
BACKGROUND
Yeast cells polarize, bud, and divide in either of two genetically programmed patterns: axial or bipolar. The Saccharomyces cerevisiae gene BUD1 (also known as RSR1) encodes a Ras-related GTPase critical for selection of a bud sites in these patterns. To distinguish between possible mechanisms of Bud1p action, we have examined the function and subcellular localization of Bud1p in a variety of mutant situations.


RESULTS
Bud1p has 57% identity to H-ras, except for an 81 amino-acid insertion near the carboxyl terminus. Mutation of the proposed BUD1 effector domain produces a protein which can neither support normal patterns of budding nor interact with CDC24, which encodes a likely Bud1p effector. A version of Bud1p deleted for the 81 amino-acid unique region is essentially wild-type. Immunofluorescence and cell fractionation indicate that Bud1p remains associated with the membrane throughout its GTPase cycle. Both potential effectors of Bud1p, Bem1p and Cdc24p, are also membrane associated even in the absence of Bud1p, suggesting that Bud1p is not required to dock these proteins from the cytosol but may couple these proteins and others within the plane of the plasma membrane.


CONCLUSIONS
Based upon observations reported here and elsewhere, we propose a novel mechanism of Bud1p GTPase action. Like Ras, Bud1p GTPase is constitutively associated with the plasma membrane; however, concentrated activities of Bud5p GDP-GTP exchange factor and Bud2p GTPase-activating protein at the future bud site promote rapid cycling of Bud1p between GTP- and GDP-bound conformations in a spatially restricted manner. Local GTPase cycling serves to efficiently nucleate complexes between polarity establishment functions that direct cytoskeletal polarization towards the bud site.
Michelitch
M
M
Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Chant
J
J
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins
D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; C084013:RSR1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins