Arrestin-mediated endocytosis of yeast plasma membrane transporters

Abstract

Many plasma membrane transporters in yeast are endocytosed in response to excess substrate or certain stresses and degraded in the vacuole. Endocytosis invariably requires ubiquitination by the HECT domain ligase Rsp5. In the cases of the manganese transporter Smf1 and the amino acid transporters Can1, Lyp1 and Mup1 it has been shown that ubiquitination is mediated by arrestin-like adaptor proteins that bind to Rsp5 and recognize specific transporters. As yeast contains a large family of arrestins, this has been suggested as a general model for transporter regulation; however, analysis is complicated by redundancy amongst the arrestins. We have tested this model by removing all the arrestins and examining the requirements for endocytosis of four more transporters, Itr1 (inositol), Hxt6 (glucose), Fur4 (uracil) and Tat2 (tryptophan). This reveals functions for the arrestins Art5/Ygr068c and Art4/Rod1, and additional roles for Art1/Ldb19, Art2/Ecm21 and Art8/Csr2. It also reveals functional redundancy between arrestins and the arrestin-like adaptors Bul1 and Bul2. In addition, we show that delivery to the vacuole often requires multiple additional ubiquitin ligases or adaptors, including the RING domain ligase Pib1, and the adaptors Bsd2, Ear1 and Ssh4, some acting redundantly. We discuss the similarities and differences in the requirements for regulation of different transporters.

Figure 1. Alignment of residues 18–51 of Ylr322c with part of the pfam consensus sequence for the arrestin N-terminal domain, with identities and similarities indicated

The full alignment is detectable by automated domain searches, but has a relatively high E-value of 0.1.

Figure 2. Itr1 degradation requires Art5

A) Confocal images of GFP-Itr1 in the indicated strains. These were grown in the absence of inositol, GFP-Itr1 synthesis induced with galactose, induction terminated with glucose, then inositol added and samples examined 0 or 60 min after this. Images are shown inverted for clarity. B) GFP-Itr1 in the 9-arrestin mutant with the indicated proteins expressed from plasmids, imaged as in A at the indicated times after the addition of inositol. C) Immunoblots of GFP-Itr1 in the indicated strains. Pgk1 was detected on the same blots as a loading control.

Figure 3. Requirements for Hxt6 degradation

A) Hxt6-GFP expressed from the TPI promoter in cells of the indicated strains, grown in raffinose then transferred to glucose for the indicated times, with cycloheximide added at the same time as the glucose. B) As A, but with no cycloheximide. Cells were in glucose for 1–2 h. C) As A, but only cycloheximide added, for 2 h. D) Immunoblots of cells treated with both glucose and cycloheximide, as indicated.

Figure 4. Requirements for Fur4 degradation

A) Images of Fur4-GFP in various strains after transient galactose induction, at the indicated times after addition of uracil or cycloheximide. B) Fur4-GFP in the indicated strains after treatment with uracil or cycloheximide for 2 h. Note that the 9-arrestin strain lacks BSD2. C) Immunoblots of Fur4-GFP as indicated.

Figure 5. Alignment of parts of the Bul1 and arrestin conserved regions, as identified by pfam

The top line in each case consists of residues 39–68 and 112–125 from a single Aspergillus clavatus protein. Residues that are similar in both the Bul1 and arrestin families are highlighted. The Bul1 sequences are all fungal; the arrestins are from a wide variety of species, and show considerable divergence.

Figure 6. Tat2 endocytosis

A) Images of Tat2-GFP in the indicated strains, induced transiently with galactose then exposed to tryptophan for the indicated times. B) As A, but endocytosis induced by treatment with cycloheximide for 2 h. C) Tat2-GFP in the 9-arrestin mutant expressing individual proteins from plasmids, as indicated, after treatment with tryptophan or cycloheximide.

Figure 7. Role of Pib1 and Bsd2

A) GFP-Smf1 in the indicated strains, at various times after addition of cadmium chloride to induce Smf1 endocytosis. Differential interference contrast (DIC) images of the final time-point are included to show that in bsd2 ear1 ssh4 cells, and in bsd2 pib1 cells, GFP-Smf1 accumulates not in vacuoles but in perivacuolar endosomes. B) Immunoblots showing less accumulation of free (vacuolar) GFP from GFP-Smf1 in the bsd2 pib1 mutant compared with bsd2. Indicated times are after the addition of cadmium chloride. C) GFP-Smf1 in pib1 cells grown in normal metal-replete medium. Under these conditions, Smf1 undergoes Bsd2-dependent transport from the Golgi to the vacuole. D) Summary of the routes taken by Smf1 and the postulated sites at which the various adaptors/ligases act. Note that Bsd2, Ear1 and Ssh4 are all membrane proteins, that pass through the Golgi to endosomes and into multivesicular bodies, and thus could act anywhere along this pathway. E) Images of cells expressing Tat2-GFP, Fur4-GFP, GFP-Itr1 or Hxt6-GFP, induced as in the previous figures, in the indicated strains. Hxt6 endocytosis was induced with glucose in the presence of cycloheximide. Note that GFP-labelled Tat2, Fur4 and Itr1 accumulate in vacuoles in pib1 and bsd2 cells, but in endosomes in bsd2 pib1 cells.

Figure 8. Summary of arrestin functions

Arrestins are represented in the middle line, transporters in the upper line (this paper) or lower line [previous work; (11,12)]. The various substances used to induce endocytosis are shown (chx, cycloheximide). Solid lines indicate function of a particular arrestin with the indicated transporter and inducing condition. Dashed lines indicate weak activity. All activities were observed with arrestins expressed from their own promoters.