Modular domains mediating speci®c protein±protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67 phox , which activates the microbicidal phagocyte NADPH oxidase. Each of these speci®cally recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40 phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67 phox , which abolishes its interaction with Cdc24p, confers on cells temperaturesensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif-containing region of p40 phox , which restores the interaction with the altered Bem1p. This domain-swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

E129,A131 , which causes a morphological abnormality, was expressed. These results provide a direct molecular basis for the action of Rho3 on exocytosis and the actin cytoskeleton.

LTE1 belongs to the CDC25 family that encodes a guanine nucleotide exchange factor for GTP-binding proteins of the ras family. Previously we have shown that LTE1 is essential for termination of M phase at low temperatures. We have identified TEMI as a gene that, when present on a multicopy plasmid, suppresses the cold-sensitive phenotype of itel. Sequence analysis of TEMI and GTP-binding analysis of the gene product revealed that TEMI encodes a novel low-molecular-weight GTP-binding protein. In eucaryotic cells, cell cycle progresses with the oscillation of activity of Cdk kinases, including M-phase-promoting factor (MPF). During M phase, MPF is activated at the onset of M phase and is inactivated at the end of this phase (24-26). The activity of MPF is dependent on its state of phosphorylation and its association with cyclins. Regulatory mechanisms working at the initiation of M phase have been clarified, whereas regulatory systems required for termination of M phase remain unclear.Cell division cycle (cdc) mutants of the budding yeast Saccharomyces cerevisiae have contributed to the identification of many components that are involved in the regulatory machinery of the cell cycle (11). Several genes, such as CDC15, CDC5, DBF2, DBF20, and CDC14, have been identified as genes working on the termination of M phase (14,15,33,42,45). Mutations in these genes arrest cells at anaphase or at telophase under restrictive conditions. CDC15, CDC5, DBF2, and DBF20 encode protein kinases, and CDC14 encodes a phosphotyrosine phosphatase, indicating that phosphorylation and dephosphorylation of proteins are required to exit from M phase. However, how the activities of these kinases and the phosphatase are controlled is obscure.Low-molecular-weight GTP-binding proteins have been established as a switching element of intracellular pathways via the conformational change between the GTP-binding state and GDP-binding state of the proteins. The transition from the GDP-binding state to the GTP-binding state is catalyzed by guanine nucleotide exchange factors for each low-molecularweight GTP-binding protein (2-4). LTE1 encodes a protein that is homologous to the CDC25 protein, which is the guanine nucleotide exchange factor for the yeast Ras proteins encoded by RAS1 and RAS2 (3,35,47 GTP-binding protein which is required for the termination of M phase, and genetic interaction of TEM1 with LTE1 and CDC15. MATERIALS AND METHODSMicrobial manipulation and analysis. The S. cerevisiae strains used in this study are RAY3A-D (MA Ta/MATa ura3/ ura3 leu2/leu2 his3/his3 trpl/trpl) (41) and E0045 (MA Ta ura3 leu2 his3 trpl ltel::HIS3) (35) and derivatives from them. Yeast cells were grown in either the rich medium yeast extractpeptone-dextrose (YPD) or the synthetic medium SC, which is SD containing appropriate auxotrophic supplements (34). SC-URA or SC-LEU was SC without uracil or leucine. YPGal was YPD but including 5% galactose and 0.2% sucrose instead of 2% glucose (21). Yeast transformations were performed by the method of Ito et al. (13), a...

We identified a novel Rho gene rho3+ and studied its interaction with diaphanous/formin for3+ in the fission yeast Schizosaccharomyces pombe. Both rho3 null cells and for3 null cells showed defects in organization of not only actin cytoskeleton but also cytoplasmic microtubules (MTs). rho3 for3double null cells had defects that were more severe than each single null cell: polarized growth was deficient in the double null cells. Function of For3 needed the highly conserved FH1 and FH2 domains, an N-terminal region containing a Rho-binding domain, and the C-terminal region. For3 bound to active forms of both Rho3 and Cdc42 but not to that of Rho1. For3 was localized as dots to the ends of interphase cells and to the mid-region in dividing cells. This localization was probably dependent on its interaction with Rho proteins. Overexpression of For3 produced huge swollen cells containing depolarized F-actin patches and thick cytoplasmic MT bundles. In addition, overexpression of a constitutively active Rho3Q71L induced a strong defect in cytokinesis. In conclusion, we propose that the Rho3-For3 signaling system functions in the polarized cell growth of fission yeast by controlling both actin cytoskeleton and MTs.

Class V myosins play a pivotal role in organelle distribution. In the budding yeast, Myo2p, a class V myosin, is essential for mitochondrial distribution. We identified MMR1 as a high-dose suppressor of the myo2 mitochondrial defect and that Mmr1p resides restrictively on the bud-localizing mitochondria and forms a complex with Myo2p tail. Mmr1p loss delayed mitochondrial transfer to buds and completely abolished mitochondrial distribution in the absence of Ypt11p, which promotes mitochondrial distribution by complex formation with Myo2p tail. The myo2-573 mutation, which causes a mitochondrial distribution defect and inactivates the Mmr1p function, reduced association between Myo2p and Mmr1p and depolarized Mmr1p localization on mitochondria. These strongly suggest that Mmr1p is a key mitochondrial component of the link between Myo2p and mitochondria for Myo2p-dependent mitochondrial distribution. Genetical analysis revealed that the Mmr1p-Myo2p pathway is independent of the Ypt11p-Myo2p pathway, suggesting that an essential system for mitochondrial distribution is composed of two independent Myo2p pathways.