Figure 1.
Figure 1. K63-linked ubiquitylation is required for MVB sorting of Gap1 and CPS. Cells expressing Ub or UbK63R as the sole source of Ub were grown in glucose- and proline-containing yeast nitrogen base medium. (A) gap1Δ cells were transformed with a centromeric vector carrying the GAP1 gene or its gap1K9R, gap1K16R, or gap1K9,16R allele. Cells were collected 5 min after the addition of 100 mM ammonium and were used to prepare total protein extracts, and the Gap1 ubiquitylation profile was examined by Western blotting using anti-Gap1 antibodies. Ubiquitylated forms of Gap1 corresponding to an additional mass of ∼7 kD are indicated with dots, whereas the molecular mass of the band marked with an asterisk suggests that it corresponds to a modified form of the upper ubiquitylated conjugate. In the case of cells expressing native Gap1 and Ub, more than two bands may be detected if Ub is overproduced or after a longer incubation in the presence of ammonium (Fig. S1). (B) gap1Δ or gap1Δ ypt6Δ cells were transformed with vectors as in A. Gap1 activity was measured before and 120 min after the addition of 100 mM ammonium. Graph bars represent the percentage of Gap1 initial activity remaining at time 120 min (mean of two independent experiments). Error bars represent standard deviation. (C and E) gap1Δ cells were transformed with a centromeric vector encoding Gap1-GFP, Gap1K9R-GFP, Gap1K16R-GFP, or Gap1K9,16R-GFP. Gap1-GFP localization was examined by fluorescence microscopy before and 120 min after the addition of 100 mM ammonium. The vacuolar membrane was labeled with the lipophilic marker FM4-64. (D) Total protein extracts were prepared from cells as in A and collected before and at different times after the addition of ammonium. Gap1 stability was analyzed by immunoblotting with anti-Gap1 antibodies. (F) The ubiquitylation profile of CPS was examined by Western blotting using anti-HA antibodies in total protein extracts prepared from CPS-HA3 cells. Ubiquitylated forms corresponding to an additional mass of ∼7 kD are indicated with dots. (G) Cells were transformed with a vector encoding GFP-CPS or Sna3-GFP. The localization of GFP-CPS and Sna3-GFP was examined by fluorescence microscopy. Bars, 5 µm.

K63-linked ubiquitylation is required for MVB sorting of Gap1 and CPS. Cells expressing Ub or UbK63R as the sole source of Ub were grown in glucose- and proline-containing yeast nitrogen base medium. (A) gap1Δ cells were transformed with a centromeric vector carrying the GAP1 gene or its gap1K9R, gap1K16R, or gap1K9,16R allele. Cells were collected 5 min after the addition of 100 mM ammonium and were used to prepare total protein extracts, and the Gap1 ubiquitylation profile was examined by Western blotting using anti-Gap1 antibodies. Ubiquitylated forms of Gap1 corresponding to an additional mass of ∼7 kD are indicated with dots, whereas the molecular mass of the band marked with an asterisk suggests that it corresponds to a modified form of the upper ubiquitylated conjugate. In the case of cells expressing native Gap1 and Ub, more than two bands may be detected if Ub is overproduced or after a longer incubation in the presence of ammonium (Fig. S1). (B) gap1Δ or gap1Δ ypt6Δ cells were transformed with vectors as in A. Gap1 activity was measured before and 120 min after the addition of 100 mM ammonium. Graph bars represent the percentage of Gap1 initial activity remaining at time 120 min (mean of two independent experiments). Error bars represent standard deviation. (C and E) gap1Δ cells were transformed with a centromeric vector encoding Gap1-GFP, Gap1K9R-GFP, Gap1K16R-GFP, or Gap1K9,16R-GFP. Gap1-GFP localization was examined by fluorescence microscopy before and 120 min after the addition of 100 mM ammonium. The vacuolar membrane was labeled with the lipophilic marker FM4-64. (D) Total protein extracts were prepared from cells as in A and collected before and at different times after the addition of ammonium. Gap1 stability was analyzed by immunoblotting with anti-Gap1 antibodies. (F) The ubiquitylation profile of CPS was examined by Western blotting using anti-HA antibodies in total protein extracts prepared from CPS-HA3 cells. Ubiquitylated forms corresponding to an additional mass of ∼7 kD are indicated with dots. (G) Cells were transformed with a vector encoding GFP-CPS or Sna3-GFP. The localization of GFP-CPS and Sna3-GFP was examined by fluorescence microscopy. Bars, 5 µm.

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