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. 2002 Apr 1;157(1):63–78. doi: 10.1083/jcb.200201037

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Copyright © 2002, The Rockefeller University Press

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Figure 4. — Kes1p PH domain mutations and PIP binding. (A) Mutations in Kes1p PH domain compromise PIP binding. Photoaffinity-labeling assays were performed with mutant Kes1ps using [³H]BZDC-PI(4,5)P₂ as photo-probe. Covalent Kes1p–photo-probe adducts were resolved by SDS-PAGE and visualized by autoradiography. Kes1p^E312K and full-length Kes1p^W317A fail to bind [³H]BZDC-PI(4,5)P₂. A proteolytic breakdown product of Kes1p^W317A (Kes1p^W317A*) regains PIP binding competence. (B) MALDI-TOF mass spectrometry of Kes1p^W317A and Kes1p^W317A*. The mass to charge ratios of full-length Kes1p^W317A and the Kes1p^W317A* truncation product are shown and the corresponding mass values for each are indicated. Below is an illustration of the Kes1p domain structure where the OSBP and PH domains are designated by the black and gray boxes, respectively. The location of the W₃₁₇A missense substitution within the PH domain is shown by a black bar, and the surrounding wild-type sequence is displayed at bottom (W corresponds to W₃₁₇). The R₃₁₄K₃₁₅ motif that defines the site of proteolysis that generates Kes1p^W317A* is underlined. (C) COOH-terminal Kes1p truncation and PIP binding. Wild-type and mutant forms of Kes1p were genetically truncated after residue K₃₁₄ to generate protein fragments consisting of otherwise wild-type primary sequence (Kes1p^1–314). GST-tagged versions of Kes1p^1–314 and Kes1p^E312K* were purified and photolabeled with [³H]BZDC-IP₃ in a mixed micelle system in the absence (T) or presence (C) of competitor PI(4,5)P₂. Both full-length protein and fragments associate with [³H]BZDC-IP₃ in a manner that is subject to displacement by PI(4,5)P₂. (D) COOH-terminal truncation of Kes1p^3E fails to rescue PIP binding. GST-tagged Kes1p and Kes1p^3E, and the corresponding COOH-terminal truncation fragments Kes1p^1–314 and Kes1p^3E* were purified were photolabeled with [³H]BZDC-IP₃ in a mixed micelle system without (T) and with (C) unlabeled PI(4,5)P₂ competitor. Full-length Kes1p^3E fails to bind photo-probe, and this failure is not rescued by Kes1p truncation. (E) PIP binding is essential for Kes1p function in vivo. Centromeric plasmids (YCp) that drive physiological levels of KES1 or kes1 ^3E expression were transformed into the kes1 strain CTY159. Transformants were streaked for isolation onto selective minimal media and incubated for 3 d at 26°C or 37°C. Complementation of kes1 defects is scored by failure of transformants to grow at 37°C.

Figure 4. — Kes1p PH domain mutations and PIP binding. (A) Mutations in Kes1p PH domain compromise PIP binding. Photoaffinity-labeling assays were performed with mutant Kes1ps using [³H]BZDC-PI(4,5)P₂ as photo-probe. Covalent Kes1p–photo-probe adducts were resolved by SDS-PAGE and visualized by autoradiography. Kes1p^E312K and full-length Kes1p^W317A fail to bind [³H]BZDC-PI(4,5)P₂. A proteolytic breakdown product of Kes1p^W317A (Kes1p^W317A*) regains PIP binding competence. (B) MALDI-TOF mass spectrometry of Kes1p^W317A and Kes1p^W317A*. The mass to charge ratios of full-length Kes1p^W317A and the Kes1p^W317A* truncation product are shown and the corresponding mass values for each are indicated. Below is an illustration of the Kes1p domain structure where the OSBP and PH domains are designated by the black and gray boxes, respectively. The location of the W₃₁₇A missense substitution within the PH domain is shown by a black bar, and the surrounding wild-type sequence is displayed at bottom (W corresponds to W₃₁₇). The R₃₁₄K₃₁₅ motif that defines the site of proteolysis that generates Kes1p^W317A* is underlined. (C) COOH-terminal Kes1p truncation and PIP binding. Wild-type and mutant forms of Kes1p were genetically truncated after residue K₃₁₄ to generate protein fragments consisting of otherwise wild-type primary sequence (Kes1p^1–314). GST-tagged versions of Kes1p^1–314 and Kes1p^E312K* were purified and photolabeled with [³H]BZDC-IP₃ in a mixed micelle system in the absence (T) or presence (C) of competitor PI(4,5)P₂. Both full-length protein and fragments associate with [³H]BZDC-IP₃ in a manner that is subject to displacement by PI(4,5)P₂. (D) COOH-terminal truncation of Kes1p^3E fails to rescue PIP binding. GST-tagged Kes1p and Kes1p^3E, and the corresponding COOH-terminal truncation fragments Kes1p^1–314 and Kes1p^3E* were purified were photolabeled with [³H]BZDC-IP₃ in a mixed micelle system without (T) and with (C) unlabeled PI(4,5)P₂ competitor. Full-length Kes1p^3E fails to bind photo-probe, and this failure is not rescued by Kes1p truncation. (E) PIP binding is essential for Kes1p function in vivo. Centromeric plasmids (YCp) that drive physiological levels of KES1 or kes1 ^3E expression were transformed into the kes1 strain CTY159. Transformants were streaked for isolation onto selective minimal media and incubated for 3 d at 26°C or 37°C. Complementation of kes1 defects is scored by failure of transformants to grow at 37°C.