Figure 2. Pex30p tubulates membrane in... | Rockefeller University Press

Figure 2.

$Figure 2. Pex30p tubulates membrane in vivo and in vitro. (A) Pex30p-Flag was purified from S. cerevisiae and analyzed by SDS-PAGE followed by staining with Coomassie blue. (B) Pex30p-Flag in LDAO was mixed with EPL, incubated with Bio-Beads for 4 h, and visualized by negative-stain EM. (C) As in B, but without EPL. (D) As in B, but without protein. Bars, 200 nm. (E) Pex30p in LDAO was subjected to sucrose density-gradient centrifugation before (top) or after incubation with EPL, a fluorescent phospholipid, and Bio-Beads to remove LDAO (bottom). Fractions were immunoblotted with an anti-Flag antibody. (F) Relative amount of fluorescent phospholipid in the fractions from the bottom panel of E (EPL + Pex30p) and an identical experiment without protein (EPL). (G) Fluorescence microscopy images of the periphery of wild-type (WT) and opi1Δ cells expressing Rtn1p-GFP (green) and ss-RFP-HDEL (red). Bar, 3 µm. (H) Quantification of the experiments in G. The relative area of ER sheets was determined from the area of ss-RFP-HDEL fluorescence that did not colocalize with Rtn1p-GFP fluorescence (mean ± SE; n = 16). M, molecular mass markers (in kD).$

Pex30p tubulates membrane in vivo and in vitro. (A) Pex30p-Flag was purified from S. cerevisiae and analyzed by SDS-PAGE followed by staining with Coomassie blue. (B) Pex30p-Flag in LDAO was mixed with EPL, incubated with Bio-Beads for 4 h, and visualized by negative-stain EM. (C) As in B, but without EPL. (D) As in B, but without protein. Bars, 200 nm. (E) Pex30p in LDAO was subjected to sucrose density-gradient centrifugation before (top) or after incubation with EPL, a fluorescent phospholipid, and Bio-Beads to remove LDAO (bottom). Fractions were immunoblotted with an anti-Flag antibody. (F) Relative amount of fluorescent phospholipid in the fractions from the bottom panel of E (EPL + Pex30p) and an identical experiment without protein (EPL). (G) Fluorescence microscopy images of the periphery of wild-type (WT) and opi1Δ cells expressing Rtn1p-GFP (green) and ss-RFP-HDEL (red). Bar, 3 µm. (H) Quantification of the experiments in G. The relative area of ER sheets was determined from the area of ss-RFP-HDEL fluorescence that did not colocalize with Rtn1p-GFP fluorescence (mean ± SE; n = 16). M, molecular mass markers (in kD).

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