Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex

Abstract

Targeted endocytosis of plasma membrane (PM) proteins allows cells to adjust their complement of membrane proteins to changing extracellular conditions. For a wide variety of PM proteins, initiation of endocytosis is triggered by ubiquitination. In yeast, arrestin-related trafficking adaptors (ARTs) enable a single ubiquitin ligase, Rsp5, to specifically and selectively target a wide range of PM proteins for ubiquitination and endocytosis. However, the mechanisms that allow ARTs to specifically recognize their appropriate substrates are unknown. We present the molecular features in the methionine permease Mup1 that are required for Art1-Rsp5-mediated ubiquitination and endocytosis. A combination of genetics, fluorescence microscopy, and biochemistry reveals three critical features that comprise an ART sorting signal in the Mup1 N-terminal cytosolic tail: 1) an extended acidic patch, 2) in close proximity to the first Mup1 transmembrane domain, and 3) close to the ubiquitinated lysines. We show that a functionally similar ART sorting signal is also required for the endocytosis of a second Art1-dependent cargo, Can1, suggesting a common mechanism for recognition of Art1 substrates. We isolate two separate suppressor mutations in the Art1 C-terminal domain that allele-specifically restore endocytosis of two Mup1 acidic patch mutants, consistent with an interaction between the Art1 C-terminus and the Mup1 acidic patch. We propose that this interaction is required for recruitment of the Art1-Rsp5 ubiquitination complex.

FIGURE 1:

Identification of mutations that block Mup1 degradation by an unbiased mutagenesis screen. (A) Principle of the mutagenesis screen. Yeast cells lacking the histidine biosynthesis enzyme His3 gain histidine prototrophy by expression of Mup1-GFP-His3. On methionine stimulation and Art1 overexpression, the construct is turned over via endocytosis, sorting, and trafficking through the MVB pathway, causing the cells to return to histidine auxotrophy. Mutations that block endocytosis prevent degradation of the chimeric construct and allow growth in the absence of histidine even upon methionine stimulation. (B) Yeast cells expressing Art1 from the Tet-Off promoter were transformed with the indicated constructs, and serial 10-fold dilutions were spotted on synthetic medium plates containing or lacking histidine and methionine, as indicated. Medium lacking histidine was supplemented with 1 mM 3-AT, an inhibitor of His3, to enhance stringency. Doxycycline was added to the medium to a final concentration of 2.5 μg/ml to shut down expression of Art1. A strain with HIS3-marked Vph1-mCherry integrated into its genome served as the HIS3+ control. (C) Wild-type cells expressing Art1 from the Tet-Off promoter and the indicated mutated variants of Mup1-GFP-His3 were grown as described in B. Plates lacking histidine contain 5 mM 3-AT. (D) Fluorescence microscopy analysis of integrated Mup1-GFP and Mup1(Q49R)-GFP in wild-type yeast cells. The cells were grown to mid log phase in synthetic medium at 30°C and imaged before (ctrl) or after (+Met) 90 min 20 μg/ml methionine treatment, as indicated. Dashed lines represent cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry. (E) Cartoon of the PM 12-TMD methionine permease Mup1 depicting the location of Q49R, G47S, and D43N mutations. (F) Yeast cells expressing GFP-tagged Mup1 or a Mup1 deletion mutant lacking residues 17–56 of the 60 amino acid–long N-tail were grown to mid log phase in synthetic medium at 30°C and subjected to fluorescence microscopy after treatment with 20 μg/ml methionine for 90 min. Dashed lines indicate cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry.

FIGURE 2:

Two regions in the Mup1 N-terminal cytosolic tail are critical for endocytic down-regulation. (A) Top, sequence of the cytosolic N-tail (residues 1–60) of the yeast methionine transporter Mup1. The two regions critical for endocytosis are underlined. Bottom, a collection of GFP-tagged Mup1 mutants was stably integrated into yeast cells expressing Vph1-mCherry as vacuolar marker. In each mutant, five consecutive residues of the N-tail were mutated to alanines. Cells were grown to mid log phase in synthetic medium at 30°C, stimulated with 20 μg/ml methionine for 90 min, and analyzed by fluorescence microscopy. Average Mup1 sorting was calculated as the ratio of vacuolar GFP to total cell GFP and normalized to wild type before (0% sorting) and after methionine stimulation (100% sorting). The numbers below each bar of the graph correspond to the position of the substituted residues within Mup1. Error bars indicate SD between fields. (B) Representative images from cells quantified in A. Cells expressing GFP-tagged Mup1 or the indicated Mup1 alanine substitutions were grown to mid log phase in synthetic medium at 30°C and subjected to fluorescence microscopy after treatment with methionine for 90 min. A complete set of images is shown in Supplemental Figure S2. Dashed lines indicate cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry. (C) Yeast cells expressing GFP-tagged Mup1 or the indicated Mup1 alanine substitutions were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. Equal volumes of culture were harvested at the indicated time points after stimulation, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a control for equal loading.

FIGURE 3:

Mup1 ubiquitination at lysines 27 and 28 initiates endocytic degradation. (A) Sequence of the N-terminal cytosolic tail of the yeast methionine transporter Mup1. Lysines are highlighted. (B) Fluorescence distribution of GFP-tagged Mup1 or Mup1 lysine mutants was analyzed in yeast cells expressing the vacuolar marker Vph1-mCherry. Cells were grown to mid log phase in synthetic medium at 30°C (control) and stimulated with 20 μg/ml methionine for 90 min (+Met). Dashed lines represent cell outlines. Scale bar, 2.5 μm. (C) Yeast cells expressing GFP-tagged Mup1 or Mup1 lysine mutants were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. Equal volumes of culture were harvested at the indicated time points after stimulation, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a loading control. (D) Yeast cells expressing GFP-tagged Mup1 were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. At the indicated time points, all cellular processes were stopped by addition of TCA to 10% to the medium. Total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a loading control. (E) Cells lacking Doa4 and coexpressing Myc-ubiquitin and Mup1-GFP or Mup1(K27,28R)-GFP were grown to mid log phase in synthetic medium at 30°C and mock treated (ctrl) or stimulated with 20 μg/ml methionine for 10 min (+Met). Native cell extracts were prepared and either analyzed by SDS–PAGE and immunoblot or used for GFP immunoprecipitation followed by SDS–PAGE and immunoblot.

FIGURE 4:

Mup1 degradation requires the acidic patch. (A) Top, yeast cells expressing Vph1-mCherry as vacuolar marker were transformed with a collection of GFP-tagged Mup1 mutants. In each mutant, one amino acid of the acidic patch was mutated to arginine. Cells were grown to mid log phase in synthetic medium at 30°C, stimulated with 20 μg/ml methionine for 90 min, and analyzed by fluorescence microscopy. Average Mup1 sorting was calculated as the ratio of vacuolar GFP to total cell GFP and normalized to wild type before (0% sorting) and after methionine stimulation (100% sorting). Error bars indicate SD between fields. Bottom, representation of the strength of the effect of each arginine substitution on Mup1 endocytosis as a “phenotype logo.” Color and size of the substituted residue indicate the group to which it belongs (weak effects are represented in light gray and small letters, and strong effects are indicated by large letters and black). (B) Fluorescence distribution of GFP-tagged Mup1 or Mup1 acidic patch mutants grown as described in A. Images of all mutants are shown in Supplemental Figure S3. Vacuolar marker: Vph1-mCherry. Scale bar, 2.5 μm. (C) Yeast cells expressing GFP-tagged Mup1 or Mup1 acidic patch mutants were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. Equal volumes of culture were harvested at the indicated time points after stimulation, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a loading control. (D) Cells lacking Doa4 and coexpressing Myc-ubiquitin and Mup1-GFP or the indicated GFP-tagged acidic patch mutants were grown to mid log phase in synthetic medium at 30°C and mock treated or stimulated with 20 μg/ml methionine for 10 min. Native cell extracts were prepared and either analyzed by SDS–PAGE and immunoblot or used for GFP immunoprecipitation followed by SDS–PAGE and immunoblot. (E) Yeast cells coexpressing RFP-tagged Mup1 (red) and the indicated GFP-tagged control or acidic patch mutants (green) were grown to mid log phase in synthetic medium at 30°C and subjected to fluorescence microscopy before (control) or after treatment with methionine for 90 min (+Met). Dashed lines indicate cell outlines. Scale bar, 2.5 μm.

FIGURE 5:

The acidic patch is position specific. (A) Experimental rationale. In the degron model (Keener and Babst, 2013), permeases mask a degradation signal (degron) in the absence of their substrates. On transport or substrate binding, the permease enters an activated state and exposes the degron, causing its own ubiquitination and degradation. Under this model, tandemizing the N-terminal 60–amino acid tail of Mup1 should cause constant exposure of the additional degron in the extra tail, since the degron-binding site of the ground-state permease is occupied by the normal tail. (B) Yeast cells expressing GFP-tagged Mup1 tandem tail constructs were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. Equal volumes of culture were harvested at the indicated time points after stimulation, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a loading control. (C) Yeast cells expressing GFP-tagged Mup1 tandem tail constructs were grown to mid log phase in synthetic medium at 30°C and subjected to fluorescence microscopy before (control) or after (+Met) treatment with methionine for 90 min. Dashed lines indicate cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry.

FIGURE 6:

Can1 contains an acidic patch. (A) Left, comparison between the N-terminal cytosolic tails of Mup1 and Can1. TMD1 (not shown) begins at 61 in Mup1 and 93 in Can1. Right, representation of Can1 and Mup1 with the position and size of the acidic patch and the ubiquitination site drawn to scale. (B) Yeast cells turn over the PM arginine permease Can1 by endocytosis and vacuolar degradation. Mutations that block Can1 endocytosis stabilize the permease at the PM, causing hypersensitivity to canavanine, a toxic analogue of arginine that enters the cells via Can1. (C) Cells lacking a chromosomal copy of Can1 were transformed with plasmids expressing either wild-type Can1 or the following point mutants and scored for growth in the presence of canavanine: E78R, Can1(E78R); 5xAla, Can1(D73A,E74A,D75A,E76A,E78A); and ARRA, Can1(D73A,E74R,D75R,E76A). The mutated region is highlighted with a bar in A. Canavanine sensitivity phenotype is scored on the right; res, resistant; sens, sensitive; wt, wild type.

FIGURE 7:

Identification of an Art1 mutant that restores endocytosis of Mup1(Q49R). (A) Yeast cells expressing GFP-tagged Mup1 acidic patch mutants and overexpressing Art1 from a single-copy plasmid and the Tet-Off promoter were grown to mid log phase in synthetic medium at 30°C, stimulated with 20 μg/ml methionine for 60 min if indicated (+Met), and subjected to fluorescence microscopy. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry. (B) Ethionine is a toxic analogue of methionine that enters the cells via the PM methionine permease Mup1. Mutants that are defective for endocytic Mup1 degradation, such as Mup1(Q49R), accumulate Mup1 at the PM and are therefore sensitive to ethionine. Mutations in Art1 that restore vacuolar trafficking of Mup1(Q49R) reduce the amount of Mup1 at the PM and cause the cells to return to an ethionine-resistant state. (C) Serial 10-fold dilutions of cells expressing hyperactivated Art1 (Art1*; Art1(79,82–85,96,99,100A); MacGurn et al., 2011) from the Tet-Off promoter were spotted on agar plates and scored for growth in the presence of ethionine. Doxycycline was added to a final concentration of 2.5 μg/ml to shut off overexpression of Art1. (D) Serial 10-fold dilutions of cells expressing Art1* or Art1*Sup from the Tet-Off promoter were spotted on agar plates and scored for growth in the absence or presence of 10 or 20 μM ethionine. Art1*Sup, Art1(79,82–85,96,99,100A,S253P,E445G,R653C). (E) Yeast cells lacking the chromosomal copy of ART1 and expressing Art1 or Art1(R563C) from a single-copy plasmid under its own promoter were transformed with the indicated variants of Mup1-GFP. The cells were grown to mid log growth phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine for 90 min. Scale bar, 2.5 μm. (F) Yeast cells lacking the chromosomal copy of ART1 and expressing Art1 or Art1(R563C) from a single-copy plasmid under its own promoter were scored for growth in the presence of canavanine.

FIGURE 8:

Multiple point mutations in Art1 allele specifically restore endocytosis of Mup1 acidic patch mutants. (A) Yeast cells expressing the indicated GFP-tagged Mup1 acidic patch mutants and carrying a single-copy plasmid overexpressing Art1, Art1(R653C), or Art1(R660D) from the Tet-Off promoter were grown to mid log growth phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine for 60 min. Dashed lines indicate cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry. (B) Cells were grown as described in A and analyzed by fluorescence microscopy. Average Mup1 sorting was calculated as the ratio of vacuolar GFP to total cell GFP and normalized to wild type before (0% sorting) and after methionine stimulation (100% sorting). Error bars indicate SD between fields. (C) Schematic representation of the Mup1 acidic patch region and the region in Art1 that harbors the suppressor mutations. Blue lines indicate which mutated residues in Art1 restore endocytosis of the respective acidic patch arginine substitution mutants in an allele-specific manner.

FIGURE 9:

Model for Art1-mediated Mup1 recognition. In the absence of methionine, Mup1 is present in a ground state, and the acidic patch is inaccessible for Art1. In the presence of methionine, however, Mup1 adopts an activated state in which the ART sorting signal becomes exposed, causing Art1-mediated recruitment of Rsp5, Mup1 ubiquitination, and trafficking to the vacuole.