A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6

Abstract

Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

Keywords: Aly1; Aly2; Arrestin; Calcineurin; Calcium; Dip5; Endocytosis; Gap1; Nutrient Permeases; Trafficking.

FIGURE 1.

Aly1 interacts with the catalytic subunit of CN from both yeast and humans. A and B, serial dilutions of PJ69-4a containing the indicated Gal4-TAD and Gal4-DBD fusions were plated on SC medium lacking the amino acids indicated and grown for 4 days at 30 °C. A, schematic depicting the region of Aly1 expressed as Gal4-TAD fusion is provided where the N-terminal arrestin-fold (N Arr), C-terminal arrestin-fold (C Arr), C-terminal tail (C Tail), and PXIXIT motif are shown in red, green, blue and yellow, respectively. B, red box helps to highlight the comparison between the full-length Aly1 and Aly1 missing the PXIXIT motif (PILKIN). C, expression of the indicated Gal4-TAD fusions in the two-hybrid reporter strain PJ69-4a was assessed by immunoblotting with anti-HA (Covance) after resolving TCA-extracted whole cell lysates (WCE) by SDS-PAGE. Red dots denote the full-length α-arrestin-Gal4-TAD fusion. White lines indicate removal of redundant replicate lanes. D, copurification of the catalytic subunit of CN, Cna1-GFP, with GST, GST-Aly1, or GST-Aly1^ΔPILKIN (expressed from pKK212-derived plasmids) from extracts of BJ5459 cells was assessed by immunoblotting. 1% of the whole cell lysates used as input for the pulldowns is shown.

FIGURE 2.

Aly1, but not Aly1^ΔPILKIN, is a CN substrate in vitro and in vivo. A, GST-tagged Aly1, Aly1^ΔPILKIN, Aly1, and Crz1 were purified on glutathione-Sepharose beads yeast extracts, incubated with [γ-³²P]ATP, and phosphorylated by copurifying kinases. Glutathione-bound proteins were washed to squelch further phosphorylation, incubated with CN-trunc or λ-phosphatase for the indicated times, and assessed by SDS-PAGE. Gels were stained for total protein or imaged to detect ³²P. Representative data from one of four replicates are shown. B, values plotted are the mean percent decrease in phospho-signal upon addition of CN-trunc (normalized for loading) for four replicate experiments performed as in A, and error bars represent the standard deviation of the means. C, BJ5459 cells expressing GST-Aly1, GST-Aly1^ΔPILKIN (a deletion of the CN-binding site), GST-Aly1^PVIVIT (a high affinity CN-binding site, Aly1^PVIVIT), or GST-Aly1^AAAAAA (mutation of the CN-binding site to alanines) were treated with nothing (−), 200 mm calcium chloride (Ca), 2 μg/ml FK506 (calcineurin inhibitor; FK), or a combination of FK506 followed by Ca (^FK/_Ca). WCEs generated by TCA extraction were resolved by on 4% acrylamide gels and assessed by immunoblotting. The black line between lanes in C indicates where samples were run on two separate gels. D, WCEs from cells treated as in C were TCA-extracted from BJ5459 cells lacking either the regulatory subunit of CN (cnb1Δ) or the two catalytic subunits of CN (cna1Δ cna2Δ). A white line indicates lane removal. E, treatment of GST-purified Aly1 and Aly1 mutants with calf intestinal alkaline phosphatase assists in identifying Aly1 phospho-species. BJ5459 cells expressing either GST-Aly1, GST-Aly1^ΔPILKIN, GST-Aly1^PVIVIT, or GST-Aly1^AAAAAA (each expressed from pKK212-derived plasmids) were treated with nothing (−), Ca, FK, or both FK and Ca, and WCEs were made by glass bead lysis. GST-tagged arrestins were purified from these lysates and treated with calf intestinal alkaline phosphatase (+ CIP) or mock incubated in CIP buffer (− CIP) at 37 °C for 30 min. Arrestins were then resolved on 4% acrylamide gels and analyzed by immunoblotting using a Li-COR Odyssey imaging station, allowing for detection of both the anti-ubiquitin (Ub) antibody (with anti-mouse IRDye 680) and the anti-GST antibody (with anti-rabbit IRDye 800) simultaneously. The white asterisks denote a proteolysis product generated during protein extraction.

FIGURE 3.

CN regulation does not alter Aly1 stability or association with Rsp5. A, BJ5459 cells expressing either GST-Aly1 or GST-Aly1^ΔPILKIN (from pKK212-derived plasmids) were treated with 200 mm calcium for 10 min (to stimulate CN activity and dephosphorylation of the WT Aly1 protein) and then incubated with 50 μg/ml CHX. Culture samples were removed at the times indicated post-CHX addition; WCEs were prepared by TCA extraction, and Aly1 mobility and levels were assessed on 4% acrylamide gels followed by immunoblotting. Representative data from three replicate experiments are shown. B, Aly1 and Aly1^ΔPILKIN protein levels (assessed as in A) were quantified, and the mean percentage of Aly1 or Aly1^ΔPILKIN protein remaining (three replicates, normalized for loading) after CHX addition is plotted with error bars representing ± S.D. C, GST, GST-Aly1, GST-Aly1^ΔPILKIN, GST-Aly1^5A, GST-Aly1^5E, GST-Aly1^Y686G, GST-Aly1^{ΔPILKIN,Y686G}, GST-Aly2, or GST-Aly2^Y703G (expressed from pKK212-derived plasmids) were extracted and purified using glutathione-Sepharose beads. Copurification of endogenous Rsp5 was assessed by immunoblotting. 4% of the WCE used as input for pulldowns is shown. A red asterisk denotes residual signal from the anti-Rsp5 antibody detection in the anti-GST pulldown blots. A horizontal white line denotes where the GST input blot has been cropped to conserve space. D, indicated GST fusions purified from cells treated with nothing (−), Ca, FK, or FK followed by Ca, resolved on 4% acrylamide gels, and immunoblotted for detection of ubiquitin (Ub) (with anti-mouse IRDye 680) and GST (anti-rabbit IRDye 800) simultaneously. The white asterisks denote a proteolysis product generated during protein extraction. For Aly1, both darker and lighter exposures are shown because the levels of Aly1 are significantly lower than those for the Y686G mutants. The darker exposure for wild-type Aly1 allows visualization of the calcineurin-dependent band shift and serves as a ubiquitinylated control for the Y686G mutant proteins. E, growth of serial dilutions of BY4741 cells containing pRS426 (vector), pRS426-Aly1, pRS426-Aly1^Y686G, pRS426-Aly1^ΔPILKIN, pRS426-Aly1^{ΔPILKIN,Y686G}, pRS426-Aly2, or pRS426-Aly1^Y703G on SC medium lacking uracil (Ura⁻ control medium) or SC-Ura⁻ with rapamycin.

FIGURE 4.

Identification of a subset of Aly1 phospho-sites regulated by calcineurin. A, map of Aly1 peptides sequences identified in the MS analysis. The single letter code for amino acids encoding Aly1 is shown with regions of the protein for which peptides were identified in each of the four MS analyses performed shown in boldface green type. The PILKIN calcineurin-docking motif is shown in red; no peptides containing the PILKIN sequence were identified in Aly1^ΔPILKIN samples. The predicted, putative 14-3-3-binding motifs in Aly1 are underlined (based on Scansite for Aly1 using a medium stringency assessment). B, schematic of the Aly1 protein with the N-terminal arrestin-fold domain (N-Arr; red), C-terminal arrestin-fold domain (C-Arr; green), C-terminal unstructured tail (Tail; blue) and approximate amino acid positions for domain boundaries (numbers along the top) shown (domains predicted using Phyre (82, 83)). The calcineurin docking-site (PXIXIT motif) is indicated in yellow. Positions of phospho-sites identified using MS analysis of Aly1 or Aly1^ΔPILKIN are shown as lines below the protein with the single letter code and sequence position provided for the amino acid. Sites regulated by calcineurin are indicated in boldface red type. C, growth of serial dilutions of BY4741 cells containing pRS426 (vector), pRS426-Aly1, pRS426-Aly1^ΔPILKIN, pRS426-Aly1^5A, or pRS426-Aly1^5E on SC medium lacking uracil (Ura⁻ control medium) or SC-Ura⁻ with rapamycin (Rapa). D, WCE from BJ5459 cells expressing GST-Aly1, GST-Aly1^5A or GST-Aly1^5E were treated with nothing (−), 200 mm calcium chloride (Ca), 2 μg/ml FK506, or a combination of FK506 followed by Ca (^FK/_Ca) were generated by TCA extraction, resolved on 4% acrylamide gels, and assessed by immunoblotting. A red asterisk (within the blot) and line denoted with Ub (adjacent to blot) are used to help identify the ubiquitinylated species of Aly1.

FIGURE 5.

CN regulation is not required for Aly1-mediated Gap1 recycling. A, growth of serial dilutions of BY4741 cells containing pRS426 (vector), pRS426-Aly1, pRS426-Aly1^ΔPILKIN, pRS426-Aly2, pRS426-Aly1^5A, or pRS426-Aly1^5E on MIN 0.5% (NH₄)₂SO₄ ± AzC. B, growth of serial dilutions of aly1Δ aly2Δ (D2–6A) cells containing pRS315 (vector), pRS315-Aly1, pRS315-Aly1^ΔPILKIN, pRS315-Aly2, pRS315-Aly1^5A, or pRS315-Aly1^5E on MIN 0.5% (NH₄)₂SO₄ ± AzC. C, BY4743 cells containing pRS426 (vector, pRS426-Aly1 or pRS426-Aly1^ΔPILKIN, pRS426-Aly1^5A, pRS426-Aly1^5E, or pRS426-Aly2) and pCKB230 (Gap1-GFP) were grown in MIN 0.5% (NH₄)₂SO₄. WCEs were resolved using SDS-PAGE, and Gap1 levels were assessed by immunoblotting. Gap1 levels relative to the vector control extract are presented below the immunoblot. D and F, prototrophic BY4741 (WT) or npr1Δ cells with pCK283 and pRS426, pRS426-Aly1, pRS426-Aly1^ΔPILKIN, or pRS426-Aly2 were assayed for [¹⁴C]citrulline uptake. The mean uptake rate for seven replicates (in C) or three replicates (in E) is shown as a percentage relative to the WT containing vector, and error bars represent ± S.D. E, growth of serial dilutions of npr1Δ cells with pRS425, pRS425-Aly1, pRS425-Aly1^ΔPILKIN, pRS425-Aly2, pRS425-Aly1^5A, or pRS425-Aly1^5E on MIN 0.5% (NH₄)₂SO₄ ± AzC.

FIGURE 6.

Calcineurin-mediated dephosphorylation of Aly1 is required for Aly1-dependent trafficking of Dip5 to the vacuole. A, WCEs from BY4741, aly1Δ, aly2Δ, or aly1Δ aly2Δ cells containing a chromosomally integrated Dip5-GFP grown in MIN +0.5% (NH₄)₂SO₄ were resolved by SDS-PAGE, and Dip5 levels were assessed by immunoblotting. Dip5 levels were quantified and are presented relative to the wild-type extract below the immunoblot. B, cells lacking both ALY1 and ALY2 containing pRS315 (vector, pRS315-Aly1, pRS315-Aly1^ΔPILKIN, pRS315-Aly1^5A, pRS315-Aly1^5E, or pRS315-Aly2) or cells lacking DIP5 were assayed for [¹⁴C]aspartic acid uptake. The mean uptake rate for a minimum of four replicates is shown as a percentage relative to aly1Δ aly2Δ with vector and error bars represent ± S.D. Unpaired Student's t tests were performed to assess the significance of these data; *** indicates a p value <0.0001. It should be noted that Aly1, Aly1^5A, and Aly2 were all significantly less than the vector control (p value <0.001) but were not significantly different from one another (p value >0.01). Similarly, Aly1^ΔPILKIN and Aly1^5E were not significantly different from one another (p value >0.01). C, Dip5-GFP in aly1Δ aly2Δ cells containing pRS315, -Aly2, -Aly1, -Aly1^ΔPILKIN, -Aly1^5A, or -Aly1^5E was visualized by fluorescence microscopy, and two panels of representative cell images are presented for each. Aly2, Aly1, and the dephosphorylation mimetic (Aly1^5A) restore endocytosis of Dip5, as evidenced by vacuolar fluorescence in >46% of cells, although cells containing vector or mutants of that mimic the phosphorylated form of Aly1 have predominantly PM-localized Dip5 (<27% of cells displayed dim vacuolar fluorescence). D, plasma membrane fluorescence intensities for Dip5-GFP (as shown in C) were normalized to background and quantified for >140 cells per strain (see “Experimental Procedures”). The mean plasma membrane intensity is plotted ± S.E. (in arbitrary units). A one-way ANOVA with Tukey's post hoc comparison was used to assess the statistical significance of fluorescence differences compared with the vector control (***, p value <0.0001; ns, not significant p value >0.01). E, ratio of plasma membrane fluorescence to vacuolar fluorescence for Dip5-GFP (as shown in C) was measured for a minimum of 40 cells per condition (see “Experimental Procedures”). A one-way ANOVA with Tukey's post hoc comparison was used to assess the statistical significance for each of these ratios compared with the pRS315 vector control (***, p value <0.0001; ** p value <0.001; *, p value <0.01, and ns = not significant p value >0.01). F, aly1Δ aly2Δ cells containing pRS315, -Aly2, -Aly1, -Aly1^ΔPILKIN, -Aly1^5A, or -Aly1^5E and a chromosomally integrated Dip5-GFP were grown in MIN +0.5% (NH₄)₂SO₄ medium, and 200 μg/ml aspartic acid and glutamic acid were added to trigger internalization and degradation of Dip5. Cells were harvested at the times indicated post-Asp/Glu addition; WCEs were prepared using TCA extraction and analyzed by SDS-PAGE and immunoblotting. Data representing one of the three replicate experiments performed are presented with the quantification for the percentage of Dip5 remaining in each lane relative to the t = 0 point and normalized for alterations in the G6PD loading control is indicated. G, Dip5-GFP band intensities from three replicate experiments (representative panel in F) were measured, normalized to the G6PD loading control, and the mean percentage of Dip5 remaining post Asp/Glu addition ± S.E. is plotted. DIC, differential interference contrast.

FIGURE 7.

CN-regulation of Aly1 does not alter Aly1-association with 14-3-3 proteins. GST, GST-Aly1, GST-Aly1^ΔPILKIN, GST-Aly1^5A, GST-Aly1^5E, or GST-Aly2 (expressed from pKK212-derived plasmids) were extracted and purified from BJ5459 cells using glutathione-Sepharose. Copurification of endogenous 14-3-3 proteins (Bmh1 is lower band and Bmh2 is upper band in doublet) was assessed by immunoblotting. 2% of the WCE used as input for pulldowns is shown.

FIGURE 8.

Model for phospho-regulation of α-arrestin-mediated trafficking. Unidentified kinases (purple rectangle) maintain Aly1 (red hexagon) in its fully phosphorylated state (phosphosites represented as gray and green circles), which is unable to stimulate internalization of the aspartic acid/glutamic acid transporter Dip5. When calcineurin (CN; blue oval) is activated by calcium, as may occur in response to excess aspartic acid/glutamic acid (indicated gray dashed line), it dephosphorylates Aly1, and this dephosphorylation at specific sites (removal of CN-regulated phosphosites; loss of green circles) is required for optimal Aly1-mediated endocytosis of Dip5. Dephosphorylated Aly1 promotes endocytosis and/or trafficking of Dip5 to the vacuole (indicated by dashed black arrow and trafficking route denoted by red line).