Figure 4.

Ubiquitination of Gap1 is impaired in npi1 cells. (A) Cells were grown on proline medium, and Gap1 activity was measured by incorporation of [¹⁴C]citrulline (0.1 mM) before (t = 0) and at several times after addition of (NH₄)₂SO₄ (10 mM) in strains 24346c (wild type; ▪) and 27038a (npi1; ○). The Gap1 activities were expressed in nanomoles per minute per milliliter. (B) Immunoblot of Gap1 from membrane-enriched cell fractions prepared before (t = 0) and at several times after addition of (NH₄)₂SO₄. The major Gap1 signal is composed of two bands at ∼60 kDa, and the positions of ubiquitinated forms are indicated with dots. (C) Quantitation of the Gap1 signal in immunoblot shown in the upper part of B (wild-type cells). The immunoblot was scanned with a densitometer to measure the amount of total and ubiquitinated Gap1 present at each time point. Each value is expressed as percentage of the Gap1 found in all forms at t = 0 min.