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Published online 2010 November 30. doi: 10.1038/msb.2010.87

Figure 7

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The 2.0 Å X-ray structure of Slm1-PH reveals two putative binding sites required for Slm1 function in vivo. (A) Comparison of the structures of Slm1-PH with that of PLCδ-PH (Protein Data Bank (PDB) entry 1mai). The two structures are represented in the same orientations. PLCδ-PH binds lipid within a positively charged canonical site conserved in Slm1 (orange circle). Slm1 presents an additional positively charged cavity (gray circle). Two key residues (R477 and R478) were identified that contribute to both positively charged binding sites in Slm1. Side chain of analogous residues in PLCδ (K30 and K32) point exclusively to the canonical binding site. Electronegative regions are colored red and electropositive regions blue. (B) Sequence alignment of Slm1-PH and PLCδ-PH. Mutated amino acids in Slm1 (R477 and R478) and their analogous in PLCδ are boxed in black. Conserved residues are blue if positively charged and green if hydrophobic. (C) The two putative Slm1-PH-binding sites are required for Slm1 membrane association with the eisosome. Kymograph representations of Slm1-GFP and Pil1-RFP (1 frame per 1.5 s). Green lines above the kymograph represent the association dynamics of Slm1-GFP with the Pil1-RFP (red lines). Mutations affecting clusters of positive charges in one (R477A or R478A) or both (R478A/R477A) resulted in specific destabilization of Slm1 association with Pil1-containing microdomains. (D) The two putative Slm1-PH-binding sites are required for Slm1 function in vivo. Strains carrying Slm1 mutations in one (R477A or R478A) or both (R477A/R478A) putative lipid-binding sites show cumulative phenotypic defects. Upper panels show growth defect in the presence of myriocin; lower panels during heat stress. The bar chart shows actin polarization defects following heat stress. * decrease for the double mutants is significant compared with wild-type (P<0.01), R477A (P<0.01) and R478A (P<0.01) single mutants. The mutations have no effect on Slm1 expression levels (western blot not shown).

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