Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane - PubMed (original) (raw)

Akr1p and the type I casein kinases act prior to the ubiquitination step of yeast endocytosis: Akr1p is required for kinase localization to the plasma membrane

Y Feng et al. Mol Cell Biol. 2000 Jul.

Abstract

Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Delta320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants. The Delta320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type alpha-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode. Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal. Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs. In akr1Delta cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation.

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Figures

FIG. 1

FIG. 1

Effects of the endocytosis-defective mutants on Ste3p constitutive ubiquitination. Receptor ubiquitination was assessed in wild-type (wt) and mutant _MAT_α GAL1-STE3 strains of two different genetic backgrounds. Strains isogenic to the wild-type strain NDY341 (left panel) include the temperature-sensitive end4-1 mutant (NDY342), as well as pep4Δ (NDY356), akr1Δ (NDY788), vrp1Δ (NDY1040), and end3Δ (NDY1046) mutants. In addition, the isogenic ste3Δ strain NDY343 was used as control for the specificity of Ste3p antibodies. Strains isogenic to NDY877 (wild-type _MAT_α GAL1-STE3) (right panel) include the yck1Δ yck2ts double mutant (NDY913) and pep4Δ (NDY1080), akr1Δ (NDY1083), and end3Δ (NDY1118) strains. Protein extracts were prepared from cell cultures, following a 2-h Ste3p expression period, induced with galactose addition to log-phase cultures growing in raffinose medium. Fifteen minutes before cells were collected for extract preparation, cultures of cells isogenic to NDY341 (left panel) or to NDY877 (right panel) were shifted from 25°C to either 37 or 30°C, respectively. Protein extracts were treated with potato acid phosphatase (see Materials and Methods) to remove heterogeneity in gel migration which results from the heterogeneous phosphorylation of Ste3p and then subjected to SDS-PAGE and Western blotting with Ste3p-specific antibodies. Arrows indicate the positions of the mono- and diubiquitinated receptors; dashes indicate the positions of proteins that cross-react with the Ste3p antibodies.

FIG. 2

FIG. 2

Turnover of an Ste3-Ub fusion protein in the endocytosis-defective mutants. A 2-h period of _GAL1_-driven expression of Ste3-Ub (A) or Ste3p (B) was induced in wild-type (wt) or end mutant yeast cultures with galactose addition and terminated with glucose addition. Fifteen minutes prior to glucose addition, cultures were shifted from 25 to 37°C. Coincident with the glucose addition (0-h time point) and at the indicated glucose chase time points, culture aliquots were removed and treated with energy poisons (see Materials and Methods). Extracts were then prepared, and the loss of the Ste3 antigen was monitored by Western blotting with Ste3p-specific antibodies. (A) Ste3-Ub turnover. Wild-type and end mutant MATα GAL1-STE3-UB strains from two different strain backgrounds were used. Strains in the end4-1 background (top panel) included wild-type (NDY990), end4-1 (NDY991), pep4Δ (NDY992), akr1Δ (NDY1011), vrp1Δ (NDY1042), and end3Δ (NDY1076) strains. NDY343 (ste3Δ) was included as control for the specificity of Ste3p antibodies. Strains in the yck1Δ yck2ts background (bottom panel) included wild-type (NDY1012), end3Δ (NDY1074), yck1Δ yck2ts (NDY1090), akr1Δ (NDY1085), akr1Δ pep4Δ (NDY1218), and yck1Δ yck2ts pep4Δ (NDY1223) strains. The high level of Ste3-Ub protein apparent in NDY992 cells (pep4Δ) likely reflects the augmented growth rate of this strain relative to the isogenic wild-type strain seen in raffinose- and/or galactose-containing culture media, not to the pep4 turnover blockade: the end mutants in which turnover is blocked (e.g., end3, end4, vrp1, yckts, and akr1 mutants) do not have similar levels of Ste3-Ub protein over accumulation. (B) Ste3p turnover in wild-type and end mutant cells. Four isogenic MATα GAL1-STE3 strains were used for this analysis, i.e., wild-type (NDY877), yck1Δ yck2ts (NDY913), akr1Δ (NDY1083), and end3Δ (NDY1118) strains.

FIG. 3

FIG. 3

Effects of akr1 and yck mutations on Ste3p phosphorylation. The constitutive and a-factor-induced phosphorylation of Ste3p was monitored by assessing the effects of a-factor treatment on the gel mobility of newly synthesized Ste3p. MATα cells were pulse-labeled for 10 min with [35S]methionine-cysteine, chased for 10 min with excess cold amino acids, and then treated for an additional 15 min with a-factor or mock treated in parallel. This protocol allows the labeled Ste3p to be maximally available at the cell surface for binding the a-factor ligand. Protein extracts were prepared from the cells, and the Ste3p purified by immune precipitation was subjected to SDS-PAGE and autoradiography. (A) Constitutive and ligand-induced phosphorylation of Ste3p. Extracts from wild-type MATα cells (NDY414), treated with or without a-factor pheromone, were either digested with potato acid phosphatase (p'ase) or mock digested (no phosphatase added). The NDY414 strain used for this experiment has Ste3p expressed from the HIS3 promoter instead of from its natural promoter. The level of Ste3p expression from the HIS3 promoter is increased two- to threefold relative to the basal-level expression from its natural promoter. The increased expression results in no obvious alteration to the profile of phosphorylated species apparent by this pulse-chase analysis (compare with panels B and C; Ste3p for these panels is expressed from its natural endogenous promoter). (B) Effects of the akr1 and yck mutations on Ste3p phosphorylation for cells growing at 30°C. Wild-type (wt) MATα cells (LRB759) as well as isogenic akr1Δ (NDY1037) and yck1Δ yck2ts (LRB757) cells were cultured at 30°C and then labeled and treated with a-factor as described above. (C) Effects of the yck mutations on Ste3p phosphorylation for cells growing at 37°C. Wild-type (LRB759) and yck1Δ yck2ts mutant (LRB757) cells cultured at 25°C were shifted to 37°C 10 min prior to the start of pulse-labeling.

FIG. 4

FIG. 4

Effects of akr1 and yck mutations on the phosphorylation of Ste3Δ320-413p. MATα GAL1-STE3Δ320-413 cells, either wild-type (wt) (NDY1039), akr1Δ (NDY1061), yck1Δ yck2ts (NDY1045), or end3Δ (NDY1070), growing in raffinose medium at 25°C were shifted to 30°C 1 h prior to the initiation of galactose-induced expression. Following a 90-min period of galactose-induced expression, cell extracts were prepared and were treated with phosphatase (p'ase) (+) or mock treated (−). Samples were then analyzed by SDS-PAGE and Western blotting with Ste3p-specific antibodies.

FIG. 5

FIG. 5

Akr1p is required together with Yck1p-Yck2p for the α-factor-induced phosphorylation of Ste2p. Cultures of three isogenic MATa strains, i.e., wild-type (wt) (LRB758), yck1Δ yck2ts (LRB756), and akr1Δ (NDY1215) strains, growing in rich medium at 25°C were shifted to 37°C for 15 min and then either treated with 10−6 M α-factor (+) for 10 min or mock treated in parallel (−). Extracts were prepared and analyzed by Western blotting with a Ste2p-specific antiserum (provided by James Konopka, SUNY at Stony Brook). Extracts prepared from the isogenic MATα strain LRB759, which does not express Ste2p, were included as a control for the specificity of the antiserum (con). The brackets at right indicate the positions of the hyperphosphorylated Ste2p species present following α-factor treatment.

FIG. 6

FIG. 6

Akr1p and Yck1p-Yck2p remain partially competent for Ste3p ligand-induced endocytosis. The a-factor-induced internalization of the Δ365 Ste3p mutant was assessed by monitoring its changing availability to added, extracellular proteases. Uptake of the Δ365 mutant was followed in four isogenic MATα GAL1-STE3Δ365 strains, i.e., wild-type (wt) (NDY1072), end3Δ (NDY1117), akr1Δ (NDY1216), and yck1Δ yck2ts (NDY662) strains. Thirty minutes following a 90-min period of galactose-induced Ste3Δ365p expression (terminated by glucose addition), cultures were shifted from 25 to 37°C (nonpermissive for the yckts mutant). Following an additional 15 min at 37°C, cultures were treated with a-factor or mock treated. At the indicated times following the start of the pheromone treatment, culture aliquots were removed and the intact cells were digested with proteases (see Materials and Methods). Extracts prepared from these cells were analyzed by Western blotting with Ste3p-specific antibodies.

FIG. 7

FIG. 7

Akr1p is required for localization of Yck2p to the plasma membrane. The wild-type strain (NDY877) or the isogenic akr1Δ strain (NDY1083) transformed by one of two plasmids carrying _GAL1_-driven YCK2 constructs N-terminally tagged by the 3xHA epitope (either pND1092, which carries the tagged wild-type Yck2p, or pND1113, which carries the equivalently tagged Yck2p mutant which has its C-terminal Cys-Cys prenylation motif replaced by Ser-Ser [CC→SS]) was used. Following a 2-h period of galactose-induced expression, cells were removed from culture and fixed, and localization of the wild-type or mutant tagged Yck2p was probed in an indirect immunofluorescence protocol using the mouse HA.11 MAb directed against the HA epitope, followed by Cy3-conjugated secondary antibody directed against mouse immunoglobulin-G. As a control for the cross-reaction of the HA.11 MAb, NDY877 cells not carrying an HA-tagged construct were fixed and processed in parallel (no HA). A Nomarski (differential interference contrast [DIC]) image of each cell grouping is shown just to the right of the fluorescent image (anti-HA).

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