Figure 6. Stt4 stabilizes Ypp1 molecules... | Rockefeller University Press

Figure 6.

$Figure 6. Stt4 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS6-Ypp1 using a Superdex 200 analytical column.$

Stt4 stabilizes Ypp1 molecules on a membrane-bound fraction of the cell. (A) Subcellular fractionation of cells expressing HA-Ypp1 (left) in a wild-type background and HA-Ypp1 distribution in the absence of SAC1 and STT4 (right). Protein component fractions in the pellet (P100; rcf of 100,000) or soluble (S100; soluble portion) fractions. Fractionation of G6PDH (soluble) and Pep13 (membrane-tethered Golgi component) are shown as controls. (B) FPLC elution profile of HA-Ypp1 isolated from the S100 portion of sac1Δ stt4Δ cells (top). Fractions were collected at the indicated elution volumes, separated by SDS-PAGE, and detected using an anti-HA antibody. The molecular mass corresponding to each elution is indicated below the blot. (C) Coimmunoprecipitation of HA-Ypp1 from yeast lysate that coexpressed GFP alone, GFP-Ypp1, or GFP-Stt4. (D) Coimmunoprecipitation of HA-Ypp1 with GFP-Ypp1 in the wild type, sac1Δ, and sac1Δ stt4Δ background. (E) FPLC sizing of E. coli–expressed and purified HIS₆-Ypp1 using a Superdex 200 analytical column.