Yeast strains used in this study.

Yeast strains used in this study.</p

Kin2’s localization to the polarity site is essential for its multicopy suppression of late secretory mutants.

Cells of strains JGY27B (sec1-1), JGY28B (sec2-41), JGY82A (sec15-1) carrying pRS426 (Vec), pRS426-KIN2, pRS426-KIN2-NT, pRS426-KIN2-N7, and pRS426-KIN2-N5 were spotted on SC-Ura plate at 1:10 serial dilution and incubated at permissive (24°C) and their respective restrictive temperatures. Pictures were taken after 4 days.</p

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

<div><p>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 <i>Saccharomyces cerevisiae</i>. 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 <i>in vivo</i>. Our study provides new insight in the localization, function, and regulation of Kin2.</p></div

Overexpression of Kin2 affects septin organization and cell wall organization.

<p><b>(A)</b> Cell morphology and Cdc3-GFP localization of cells overexpressing Kin2. Cells of strain YEF473A (WT) and YEF1238 (<i>gin4</i>Δ) with integrated <i>CDC3-GFP</i>:<i>LEU2</i> carrying pEGKT316 (Vec) or pEGKT316-KIN2 were grown on SRG-Ura plate at 30°C for 20 hr before imaging. <b>(B)</b> Morphology of wild-type and <i>swe1</i>Δ cells overexpressing Kin2. Cells of strain YEF473A (WT) and JGY2030 (<i>swe1</i>Δ) carrying pEGKT316 (Vec) or pEGKT316-KIN2 were grown on SRG-Ura plate. <b>(C)</b> Cells overexpressing Kin2 or Kin2-Δ42 are sensitive to calcofluor white and SDS. YEF473A cells carrying pEGKT316 (Vec), pEGKT316-KIN2, pEGKT316-KIN2-Δ42 plasmids were grown on SRG-Ura medium (control), SRG-Ura medium with 5 μg/ml calcofluor white, and SRG-Ura medium with 40 μg/ml SDS at 30°C. Pictures were taken after 6 days. <b>(D)</b> Chitin distribution and GFP-Ras2 localization. Cells of strain YEF473A carrying plasmids pRS315-GFP-RAS2/pEGKT316 (Vec) or pRS315-GFP-RAS2/pEGKT316-KIN2 were grown on SRG-Leu-Ura plate at 30°C for 20 hr. Chitin was stained before imaging. Bars, 5 μm.</p

Phenotypes of cells overexpressing Kin2 segments.

<p><b>(A)</b> Schematic representation of the Kin2 segments used in the study. <b>(B)</b> Cells of strain YEF473A carrying pEGKT316 (Vec), pEGKT316-KIN2, and pEGKT316-KIN2 segments were spotted on SC-Ura (Dex) plate and SRG-Ura (Gal) plate at 1:10 serial dilution and incubated at 30°C. Pictures were taken after 4 days. <b>(C)</b> Cells of strain YEF473A (WT) and YEF1238 (<i>gin4</i>Δ) carrying pEGKT316 (Vec), pEGKT316-KIN2, and pEGKT316-KIN2 segments were grown on SRG-Ura plate at 30°C for 20 hr before imaging. <b>(D-E)</b> Percentages of cells with elongated buds <b>(D)</b> and multibudded cells <b>(E)</b> in the population of budded <i>gin4</i>Δ cells overexpressing <i>KIN2</i> and <i>KIN2</i> segments as in <b>(C)</b> were quantitated. Bar, 5 μm.</p

Kin2 interacts with Rho3 and Bmh1.

<p><b>(A)</b> Two-hybrid assay of the interaction between Kin2-C1 and Rho3. The assay was performed as described in <i>Materials and Methods</i>. pGBDU-C1 (DBD, vector), pGBDU-RHO3^{Q74L, ΔC}, and pGBDU-RHO3^{T30N, ΔC} were used to pair with pOAD (AD, vector) and pOAD-KIN2-C1. Cells were grown on SC-Leu-Ura-Ade plate at 30°C for 3 days. <b>(B)</b> BiFC assay between Kin2-C9 and Rho3. Cells of strain JGY3088 (<i>kin1</i>Δ <i>kin2</i>Δ) carrying pVN1-KIN2-C9/pVC1, pVN1-KIN2-C9/pVC1-PMT-RHO3^Q74L, and pVN1/ pVC1-PMT-RHO3^Q74L pairs were grown on SC-His-Ura plate at 30°C for 16 hr. Green fluorescence was examined. Bar, 5 μm. <b>(C)</b> Two-hybrid assay of the interaction between Kin2 segments and Bmh1. pGBDU-C1 (DBD, vector) and pGBDU-KIN2 segments were used to pair with pOAD (AD, vector) and pOAD-BMH1^5-267. Cells were grown on SC-Leu-Ura-His containing 3 mM 3-AT (-His+3-AT) and SC-Leu-Ura-Ade (-Ade) plates at 30°C for 3 days.</p

Kin2 contains two distinct targeting domains for localization.

<p><b>(A)</b> Schematic representation of Kin2’s domains and the Kin2 segments used in the study. <b>(B)</b> Localization of GFP-Kin2 and GFP-Kin2 segments. Cells of strain YEF473A carrying pUG36-KIN2 and pUG36-KIN2 segments were grown on SC-Ura plate at 30°C for 16 hr and then examined for GFP fluorescence. Bar, 5 μm.</p

GFP-Kin2 localization during bud development.

<p>Cells of yeast strain YEF473A carrying plasmid pKG21-KIN2 were grown on SC-Ura plate at 30°C for 16 hr and examined for GFP fluorescence. Bar, 5 μm.</p

Kin2 interacts with Cdc11 and Pea2 via its C-terminal region.

<p><b>(A)</b> Two-hybrid assay of the interaction of Kin2 segments with Cdc11 and Pea2. The assay was performed as described in <i>Materials and Methods</i>. pGBDU-C1 (DBD, vector) and pGBDU-KIN2 segments were used to pair with pOAD (AD, vector), pOAD-CDC11^72-415 and pGAD-PEA2. Cells were grown on SC-Leu-Ura-Ade (-Ade) and SC-Leu-Ura-His (-His) plates at 30°C for 3 days. Growth indicates interaction between the DBD and AD fusion proteins. The domain structure of Cdc11 and Cdc11^72-415 segment was depicted in the upper panel. <b>(B)</b> BiFC assay between Kin2-C6 and Pea2. Cells of strain JGY3088 (<i>kin1</i>Δ <i>kin2</i>Δ) carrying pVN1-PEA2/pVC1, pVN1-PEA2/pVC1-KIN2-C6, and pVN1/pVC1-KIN2-C6 pairs were grown on SC-His-Ura plate at 30°C for 16 hr. Green fluorescence was examined by fluorescence microscopy. Bar, 5 μm.</p

A model for the functional mechanism of Kin2 in septin organization and regulation of Kin2 in the cells.

<p><b>(A)</b> Kin2 may play a role in septin organization by interacting with Cdc11 (a septin subunit) and Pea2 (a polarisome component). <b>(B)</b> Possible regulation of Kin2. Kin2 is normally in an inactive conformation as the N-terminal region (NT) and C-terminal region (CT) bind each other. The binding of protein X (such as Rho3 or Pea2) to Kin2-CT may help relieve the intramolecular inhibition. The binding of Bmh1 to Kin2-NT may help maintain Kin2 in the active conformation.</p