Site-directed Mutagenesis of the Cysteine Residues in the Neurospora crassa Plasma Membrane H-ATPase

A high-yield yeast expression system for site-directed mutagenesis of the Neurospora crassa plasma membrane H(+)-ATPase has recently been reported (Mahanty, S. K., Rao, U. S., Nicholas, R. A., and Scarborough, G. A. (1994) J. Biol. Chem. 269, 17705-17712). Using this system, each of the eight cysteine residues in the ATPase was changed to a serine or an alanine residue, producing strains C148S and C148A, C376S and C376A, C409S and C409A, C472S and C472A, C532S and C532A, C545S and C545A, C840S and C840A, and C869S and C869A, respectively. With the exception of C376S and C532S, all of the mutant ATPases are able to support the growth of yeast cells to different extents, indicating that they are functional. The C376S and C532S enzymes appear to be non-functional. After solubilization of the functional mutant ATPase molecules from isolated membranes with lysolecithin, all behaved similar to the native enzyme when subjected to glycerol density gradient centrifugation, indicating that they fold in a natural manner. The kinetic properties of these mutant enzymes were also similar to the native ATPase with the exception of C409A, which has a substantially higher Km. These results clearly indicate that none of the eight cysteine residues in the H(+)-ATPase molecule are essential for ATPase activity, but that Cys376, Cys409, and Cys532 may be in or near important sites. They also demonstrate that the previously described disulfide bridge between Cys148 and Cys840 or Cys869 plays no obvious role in the structure or function of this membrane transport enzyme

Nuclear Shuttling of Yeast Scaffold Ste5 Is Required for Its Recruitment to the Plasma Membrane and Activation of the Mating MAPK Cascade

AbstractLocalization of Ste5 to Gβ at the plasma membrane is essential for transmission of the pheromone signal to associated MAP kinase cascade enzymes. Here, we show that this crucial localization requires prior shuttling of Ste5 through the nucleus. Ste5 shuttles through the nucleus constitutively during vegetative growth. Pheromone enhances nuclear export of Ste5, and this pool translocates vectorially to the cell periphery. Remarkably, Ste5 that cannot transit the nucleus is unable to localize at the periphery and activate the pathway, while Ste5 with enhanced transit through the nucleus has enhanced ability to localize to the periphery and activate the pathway. This novel regulatory scheme may ensure that cytoplasmic Ste5 does not activate downstream kinases in the absence of pheromone and could be applicable to other membrane-recruited signaling proteins

Mapping regions in Ste5 that support Msn5-dependent and -independent nuclear export

Careful control of the available pool of the MAPK scaffold Ste5 is important for mating pathway activation and prevention of inappropriate mating differentiation in haploid S. cerevisiae. Ste5 shuttles constitutively through the nucleus where it is degraded by a ubiquitin-dependent mechanism triggered by G1 CDK phosphorylation. Here we narrow down regions of Ste5 that mediate nuclear export. Four regions in Ste5 relocalize SV40-TAgNLS-GFP-GFP from nucleus to cytoplasm. One region is N-terminal, dependent on exportin Msn5/Ste21/Kap142, and interacts with Msn5 in two hybrid assays independently of mating pheromone, Fus3, Kss1, Ptc1, the NLS/PM, and RING-H2. A second region overlaps the PH domain and Ste11 binding site and two others are on the vWA domain and include residues essential for MAPK activation. We find no evidence for dependence on Crm1/Xpo1 despite numerous potential nuclear export sequences (NESs) detected by LocNES and NETNES1.1 predictors. Thus, Msn5 (homolog of human Exportin-5) and one or more exportins or adapter molecules besides Crm1/Xpo1 may regulate Ste5 through multiple recognition sites.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author