Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae

Abstract

Addition of ammonium ions to yeast cells growing on proline as the sole nitrogen source induces rapid inactivation and degradation of the general amino acid permease Gap1 through a process requiring the Npi1/Rsp5 ubiquitin (Ub) ligase. In this study, we show that NH4+ induces endocytosis of Gap1, which is then delivered into the vacuole where it is degraded. This down-regulation is accompanied by increased conversion of Gap1 to ubiquitinated forms. Ubiquitination and subsequent degradation of Gap1 are impaired in the npi1 strain. In this mutant, the amount of Npi1/Rsp5 Ub ligase is reduced >10-fold compared with wild-type cells. The C-terminal tail of Gap1 contains sequences, including a di-leucine motif, which are required for NH4+-induced internalization and degradation of the permease. We show here that mutant Gap1 permeases affected in these sequences still bind Ub. Furthermore, we provide evidence that only a small fraction of Gap1 is modified by Ub after addition of NH4+ to mutants defective in endocytosis.

Figure 1

Ammonium-induced down-regulation of Gap1 is impaired in act1-1 mutant cells. (A) Cells were grown on proline medium, and Gap1 activity was assayed by measuring incorporation of [¹⁴C]citrulline (0.1 mM) before (t = 0) and at various times after addition of (NH₄⁺)₂SO₄ (10 mM) in strains NY13 (wild type; ○), NY279 (act1-1; •), and 24346c (wild type derived from Σ1278b; ▪). The Gap1 activities were calculated in nanomoles per minute per milliliter to avoid dilution effect due to NH₄⁺-triggered arrest of Gap1 synthesis. In proline-grown cells, initial Gap1 activity is lower in act1-1 cells than in isogenic wild-type cells (1.3 vs. 4.2 nmol · min⁻¹ · ml⁻¹). (B) Immunoblot of Gap1 from membrane-enriched cell fractions prepared before (t = 0) and at several times after addition of (NH₄)₂SO₄. Note that the disapearance of Gap1 signal in the NY13 wild-type strain is slower compared with the wild type derived from Σ1278b.

Figure 2

Ammonium-induced degradation of Gap1 is dependent on vacuolar proteases. (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 RTY1 (pep4Δ; •) and 27061b (wild type; □). The Gap1 activities were calculated in nanomoles per minute per milliliter. (B) Immunoblot of Gap1 in crude extracts prepared before (t = 0) and at several times after addition of (NH₄)₂SO₄.

Figure 3

The amount of Npi1 Ub ligase is reduced in npi1 cells. Crude extracts were prepared from cells grown on proline medium, resolved by electrophoresis, and blotted onto a nitrocellulose membrane. The blot was probed with polyclonal Nedd4 antibodies (Kumar et al. 1997). The strains used were 24346c (wild type; lane 1), 27038a (npi1; lane 2), and 27038a (npi1) transformed with plasmid YEpJYS-2 carrying the NPI1 gene (lane 3). The signal corresponding to Ub ligase Npi1/Rsp5 is marked with an asterisk.

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.

Figure 5

Effect of Ub and Ub-myc expression on the ubiquitination profile of Gap1. Cells of strain 27061b (wild type) bearing plasmid YEp96 (Ub; lanes 1 and 3) or YEp105 (Ub-myc; lanes 2 and 4) were grown in the presence of CuSO₄ to induce synthesis of Ub and Ub-myc from the CUP1 promoter. Two hours after induction, membrane-enriched cell fractions were prepared before (lanes 1 and 2) and 5 min after addition of ammonium (lanes 3 and 4) and subjected to Western analysis with anti-Gap1 antibodies. The positions of the ubiquitinated forms of Gap1 are indicated with dots, and the band marked with an arrow corresponds to the H⁺-ATPase Pma1 used as an internal control.

Figure 6

Sequence of the C-terminal region (residue 533–601) of the Gap1 permease and mutant derivatives. Arrows indicate the positions of the amino acid substitutions (shown in bold). The deleted region is indicated by joined lines.

Figure 7

Ubiquitination of C-terminal Gap1 mutants defective in down-regulation. (A) Cells were grown on proline medium, and Gap1 activity was measured by incorporation of [¹⁴C]citrulline before (t = 0) and at several times after addition of (NH₄)₂SO₄ (10 mM) in strain JOD0097 (gap1Δ) expressing the wild-type Gap1 permease (▪) or one of the three mutant permeases, Gap1^pgr (○), Gap1^LL→AA (♦), or Gap1Δ2 (•). The Gap1 activities were expressed in nanomoles per minute per milliliter. (B) Immunoblot of Gap1 from crude extracts prepared before (t = 0) and several times after (NH₄)₂SO₄ addition. (C) Immunoblot of Gap1 from membrane-enriched fractions of cells prepared before (t = 0) and 15 and 30 min after addition of (NH₄)₂SO₄. (D) Quantitation of blots shown in C. Measure of the amount of Gap1 present at each time point. Each value is expressed as a percentage of the total Gap1 found in all forms at (t = 0). Closed bars, all forms of Gap1; open bars, unconjugated forms of Gap1; stippled bars, ubiquitinated forms of Gap1.