The Sur7/PalI family transmembrane protein Tos7 (Yol019w) plays a role in secretion in budding yeast.

Tos7 (Yol019w) is a Sur7/PalI family transmembrane protein in the budding yeast Saccharomyces cerevisiae. Since the deletion of TOS7 did not affect growth or cell morphology, the cellular roles of Tos7 have not been established previously. Here, we show that high-copy TOS7 expression suppressed the growth defect of the secretion-defective RGA1-C term-overexpressing mutant and sec15-1 mutant. Moreover, Tos7 physically interacted with Boi2 and the Rho GTPase Rho3, two key regulators of exocyst assembly, suggesting that Tos7 plays a role in secretion. We also show that the deletion of TOS7 rendered the cells more sensitive to the cell wall-disrupting agents Congo red and calcofluor white while high-copy TOS7 expression had an opposite effect, suggesting that Tos7 affects cell wall organization. Finally, we show that Tos7 localized to punctate patches on the plasma membrane that were largely co-localized with the plasma membrane microdomains named MCC (membrane compartment of Can1). Together, these results suggest that Tos7 contributes to cell surface-related functions. Tos7 is likely an auxiliary component of MCC/eisosome that specifically interacts with the secretory pathway.

Roles of the PH, coiled-coil and SAM domains of the yeast polarity protein Boi2 in polarity-site localization and function in polarized growth.

Boi1 and Boi2 are paralogous proteins essential for bud formation in budding yeast. So far, the domains that target Boi1/Boi2 to the polarity sites and function in bud formation are not well understood. Here, we report that a coiled-coil domain of Boi2 cooperates with the adjacent PH domain to confer Boi2's bud-cortex localization and major function in cell growth. The PH domain portion of the PH-CC bi-domain interacts with the Rho GTPases Cdc42 and Rho3 and both interactions are independent of the GTP/GDP-bound state of each GTPase. Interestingly, high-copy RHO3 and BOI2 but not CDC42 suppressed the growth defect of RGA1-C538 overexpression and the sec15-1 mutant and this BOI2 function depends on RHO3, suggesting that Boi2 may function in the Rho3 pathway. The SAM domain of Boi2 plays an essential role in high-copy suppression of the two mutants as well as in the early bud-neck localization of Boi2. The SAM domain and the CC domain also interact homotypically. They are likely involved in the formation of Boi2-containing protein complex. Our results provide new insights in the localization and function of Boi2 and highlight the importance of the PH-CC bi-domain and the SAM domain in Boi2's localization and function.

Roles of an N-terminal coiled-coil-containing domain in the localization and function of Bem3, a Rho GTPase-activating protein in budding yeast.

GTPase-activating proteins (GAPs) play critical roles in the spatial and temporal control of small GTPases. The budding yeast Bem3 is a GAP for Cdc42, a Rho GTPase crucial for actin and septin organization. Bem3 localizes to the sites of polarized growth. However, the amino acid sequence determinants mediating recruitment of Bem3 to its physiological sites of action and those important for Bem3 function are not clear. Here, we show that Bem3's localization is guided by two distinct targeting regions-the PX-PH-domain-containing TD1 and the coiled-coil-containing TD2. TD2 localization is largely mediated by its interaction with the polarisome component Epo1 via heterotypic coiled-coil interaction. This finding reveals a novel role for the polarisome in linking Bem3 to its functional target, Cdc42. We also show that the coiled-coil domain of Bem3 interacts homotypically and this interaction is important for the regulation of Cdc42 by Bem3. Moreover, we show that overexpression of a longer version of the TD2 domain disrupts septin-ring assembly in a RhoGAP-independent manner, suggesting that TD2 may be capable of interacting with proteins implicated in septin-ring assembly. Furthermore, we show that the longer version of TD2 interacts with Kss1, a MAPK involved in filamentous growth. Kss1 is reported to localize mainly in the nucleus. We find that Kss1 also localizes to the sites of polarized growth and Bem3 interacts with Kss1 at the septin-ring assembly site. Our study provides new insights in Bem3's localization and function.

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases, Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall.

MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains-TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.