Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gβγ dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site

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

Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) pathway in Saccharomyces cerevisiae.

Ste5 is a prototype of scaffold proteins that regulate activation of mitogen-activated protein kinase (MAPK) cascades in all eukaryotes. Ste5 associates with many proteins including Gβγ (Ste4), Ste11 MAPKKK, Ste7 MAPKK, Fus3 and Kss1 MAPKs, Bem1, Cdc24. Here we show that Ste5 also associates with heat shock protein 70 chaperone (Hsp70) Ssa1 and that Ssa1 and its ortholog Ssa2 are together important for Ste5 function and efficient mating responses. The majority of purified overexpressed Ste5 associates with Ssa1. Loss of Ssa1 and Ssa2 has deleterious effects on Ste5 abundance, integrity, and localization particularly when Ste5 is expressed at native levels. The status of Ssa1 and Ssa2 influences Ste5 electrophoresis mobility and formation of high molecular weight species thought to be phosphorylated, ubiquitinylated and aggregated and lower molecular weight fragments. A Ste5 VWA domain mutant with greater propensity to form punctate foci has reduced predicted propensity to bind Ssa1 near the mutation sites and forms more punctate foci when Ssa1 Is overexpressed, supporting a dynamic protein quality control relationship between Ste5 and Ssa1. Loss of Ssa1 and Ssa2 reduces activation of Fus3 and Kss1 MAPKs and FUS1 gene expression and impairs mating shmoo morphogenesis. Surprisingly, ssa1, ssa2, ssa3 and ssa4 single, double and triple mutants can still mate, suggesting compensatory mechanisms exist for folding. Additional analysis suggests Ssa1 is the major Hsp70 chaperone for the mating and invasive growth pathways and reveals several Hsp70-Hsp90 chaperone-network proteins required for mating morphogenesis

Counteractive Control of Polarized Morphogenesis during Mating by Mitogen-activated Protein Kinase Fus3 and G1 Cyclin-dependent Kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis