Control of Ste6 recycling by ubiquitination in the early endocytic pathway in yeast

Abstract

We present evidence that ubiquitination controls sorting of the ABC-transporter Ste6 in the early endocytic pathway. The intracellular distribution of Ste6 variants with reduced ubiquitination was examined. In contrast to wild-type Ste6, which was mainly localized to internal structures, these variants accumulated at the cell surface in a polar manner. When endocytic recycling was blocked by Ypt6 inactivation, the ubiquitination deficient variants were trapped inside the cell. This indicates that the polar distribution is maintained dynamically through endocytic recycling and localized exocytosis ("kinetic polarization"). Ste6 does not appear to recycle through late endosomes, because recycling was not blocked in class E vps (vacuolar protein sorting) mutants (Deltavps4, Deltavps27), which are affected in late endosome function and in the retromer mutant Deltavps35. Instead, recycling was partially affected in the sorting nexin mutant Deltasnx4, which serves as an indication that Ste6 recycles through early endosomes. Enhanced recycling of wild-type Ste6 was observed in class D vps mutants (Deltapep12, Deltavps8, and Deltavps21). The identification of putative recycling signals in Ste6 suggests that recycling is a signal-mediated process. Endocytic recycling and localized exocytosis could be important for Ste6 polarization during the mating process.

Figure 1.

Polar cell surface localization of Ste6 ΔA-box. Different yeast strains were transformed with pRK873 coding for the c-myc–tagged Ste6 ΔA-box variant. The intracellular distribution of Ste6 ΔA-box was examined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies in strains grown at 25°C (left columns) or shifted to 37°C (right columns) for 20 min (JD52, RKY2057, RKY2116, RKY2118) or 60 min (RKY1718, RKY2113). (A) JD52 (WT), (B) RKY1718 (cdc12-6), (C) RKY2057 (ypt6-2), (D) RKY2113 (sec14), (E) RKY2118 (end4 ypt6-2), and (F) RKY2116 (end4 Δsnx4). Alternating: FITC fluorescence and phase contrast images.

Figure 2.

Phenotypes of the Ste6 R¹¹ variant. (A) Mutagenesis of the Ste6 linker-region. Based on the distribution of charged amino acids (indicated by + or –), the Ste6 linker-region can be divided into an acidic part (A-box) and a basic part (B-box). Changes introduced by site-directed mutagenesis are indicated: short arrows: lysine to arginine mutations in the Ste6 R¹¹ variant; long arrows: F/Y mutations (see also Table 2). (B) Ste6 R¹¹ ubiquitination. Ste6 was immunoprecipitated from cell extracts prepared from the Δste6 strain RKY959 transformed with a plasmid expressing HA-tagged ubiquitin (YEp112; Hochstrasser et al., 1991) and (1) YEplac195 (vector), (2) pRK69 (2 μ-STE6), or (3) pRK814 (2 μ-STE6 R¹¹). The immunoprecipitates were analyzed by Western blotting with anti-Ste6 antibodies (left) and anti-HA antibodies (right). Expression of HA-ubiquitin from the CUP1 promoter of YEp112 was induced with 0.5 mM CuSO₄ 3 h before extract preparation. Arrows indicate mono- and di-ubiquitinated forms of Ste6. (C) The Ste6 half-life was determined by a pulse-chase experiment. Cells of the Δste6 strain RKY959 transformed with pRK278 (WT Ste6, left) or pRK658 (Ste6 R¹¹, right) were labeled with ³⁵S-Translabel (ICN Biomedicals, Costa Mesa, CA) for 15 min. Ste6 was immunoprecipitated from cell extracts prepared after different chase periods (as indicated) and examined by autoradiography. An arrow indicates the Ste6 band; background bands are marked with an asterisk.

Figure 3.

Fusion to ubiquitin suppresses the Ste6 R¹¹ recycling phenotype. (A) The STE6 deletion strain RKY959 was transformed with different Ste6 encoding plasmids; from top to bottom: pYKS2 (WT Ste6), pRK659 (Ste6 R¹¹), pRK1043 (Ste6 R¹¹-Ub). The distribution of the Ste6 variants was examined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies; left, FITC fluorescence; right, phase contrast image. (B) Equal amounts of cell extract were examined for the presence of the different Ste6 variants by Western blotting with anti-Ste6 antibodies: (1) pYKS2 (WT Ste6), (2) pRK659 (Ste6 R¹¹), and (3) pRK1043 (Ste6 R¹¹-Ub).

Figure 4.

Ste6 distribution in different protein sorting mutants. Different mutant strains were transformed with pYKS2 (WT Ste6, left row), pRK264 (Ste6 ΔA-box, middle row), or pRK659 (Ste6 R¹¹, right row). The Ste6 distribution was examined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies. The strains used are (from top to bottom): JD52 (WT), RKY1511 (Δvps4), RKY1876 (Δvps27), RKY2074 (Δvps35), RKY1634 (Δsnx4), and RKY1875 (Δvps8).

Figure 5.

Effect of class D vps mutants on Ste6 trafficking. (A) Epistasis analysis: single (left row) or double mutants (right row) were transformed with pYKS2 (WT Ste6) and the Ste6 distribution was determined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies. The following strains were used (from top to bottom): RKY1920 (Δvps21)/RKY1926 (Δvps8 Δvps21), RKY1921 (Δpep12)/RKY1930(Δvps8 Δpep12), RKY1922 (Δbro1)/RKY1928 (Δvps8 Δbro1), and RKY1510 (Δsnf7)/RKY1927 (Δvps8 Δsnf7). (B) Ste6 was immunoprecipitated from cell extracts of strains transformed with a plasmid expressing HA-tagged ubiquitin (YEp112; Hochstrasser et al., 1991) and YEplac195 (1) or pRK69 (2 μ-STE6; 2 and 3). The following strains were used: (1) RKY959 (Δste6), (2) JD52 (WT), and (3) RKY1875 (Δvps8). The immunoprecipitates were analyzed by Western blotting with anti-Ste6 antibodies (left) and anti-HA antibodies (right). Expression of HA-ubiquitin from the CUP1 promoter of YEp112 was induced with 0.5 mM CuSO₄ 3 h before extract preparation. A background band is marked by an asterisk; ubiquitinated Ste6 forms are marked by arrows.

Figure 6.

Identification of putative recycling signals in Ste6. Aromatic amino acids in the Ste6 linker-region were mutated to leucine (Figure 2A, Table 2). The mutated plasmids were transformed into the Δvps8 strain RKY1875. The following plasmids were used (from top to bottom): pYKS2 (WT Ste6), pRK888 (Ste6 F630L), pRK889 (Ste6 Y648L), pRK890 (Ste6 F656L/Y657L/Y661L), pRK891 (Ste6 Y681L), and pRK892 (Ste6 Y713L). Left row, FITC fluorescence; right row, phase contrast image.

Figure 7.

Trafficking of the Ste6* Y681L mutant. Different strains were transformed with pRK891 (Ste6* Y681L). The Ste6 distribution was examined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies. The following strains were used (from top to bottom): JD52 (WT), RKY592 (end4), and RKY1876 (Δvps27).

Figure 8.

Effect of the Y681L mutation on mating. (A) Cultures of strain JD52 transformed with pYKS2 (Ste6*, top panels) or pRK891 (Ste6* Y681L, bottom panels) were treated with α-factor (5 μM) for 2 h. Then the Ste6 distribution was determined by immunofluorescence microscopy with anti-myc primary antibodies (9E10) and FITC-conjugated anti-mouse secondary antibodies. Left, FITC fluorescence; right, phase-contrast images. (B) To examine the effect of the Y681L mutation on mating activity, the Δste6 strain RKY959 was transformed with single-copy plasmids expressing different Ste6 variants. Tenfold serial dilutions of the different cultures were spotted onto a lawn of a MATα strain. The cells were allowed to mate for 12 h and were then replica plated to a selective plate (SD minimal medium) to select for zygotes. Left panel, cell spotted on rich medium plate (YPD); middle panel, SD plate; right panel, halo-assay, culture supernatants (2-fold serial dilutions) were spotted onto a lawn of a-factor supersensitive MATα cells. Plates were incubated for 3 d at 30°C. The following plasmids were used (from top to bottom): YEplac195 (vector, Δste6), pRK278 (WT Ste6), pRK909 (Ste6 Y681L), pRK940 (Ste6*), and pRK939 (Ste6* Y681L).

Figure 9.

Sorting in the early endocytic pathway. A model for ubiquitination-dependent sorting of Ste6 in the early endocytic pathway is presented (modified from Maxfield and McGraw, 2004). Nonubiquitinated Ste6 (⋄) is sorted into tubules that pinch off from sorting endosomes giving rise to recycling endosomes. From there it can be transported to the cell surface. Ubiquitinated Ste6 (♦) is retained in the vacuolar part of the sorting endosome and directed into the multivesicular bodies (MVB) degradation pathway.