Glucose-induced ubiquitylation and endocytosis of the yeast Jen1 transporter: role of lysine 63-linked ubiquitin chains

Abstract

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys(63) linked chain(s), and Jen1-Lys(338) is one of the target residues. Ubiquitin-Lys(63)-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys(63)-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.

FIGURE 1.

Endocytosis of Jen1-HA at restrictive temperature in Casein Kinase I (yck-ts) mutant cells. A, protein extracts from SP7 cells harboring chromosomally encoded Jen1-HA and induced for the production of Jen1-HA in lactic acid for 4 h were incubated at 37 °C for 1 h in the presence (+) or absence (−) of 50 units of calf intestinal phosphatase (CIP). The samples were then separated by SDS-PAGE and analyzed for Jen1-HA by Western immunoblotting with an anti-HA antibody. The sizes of molecular weight markers are indicated. B, parental SP5 (YCK) and SP6 (yck) cells harboring chromosomally encoded Jen1-HA were induced for the production of Jen1-HA in lactic acid for 3.5 h at permissive temperature (23 °C), followed by 30 min at restrictive temperature (37 °C). Lactic acid uptake was measured at the times indicated after the addition of glucose (2%). Results are percentages of initial activities. □, wild type; ▲, yck cells. C, protein extracts were prepared at the same time points and analyzed for Jen1-HA by Western immunoblotting with an anti-HA antibody. Blots were reprobed with an anti-phosphoglycerol kinase (PGK) antibody to provide loading controls.

FIGURE 2.

Endocytosis of Jen1-HA in rsp5/npi1 mutant cells. Parental SP1 (WT) and SP2 (rsp5/npi1) cells harboring chromosomally encoded Jen1-HA were induced for 4 h in lactic acid before glucose addition. A, lactic acid uptake was measured at the times indicated after the addition of glucose (2%). The results are percentages of initial activities. □, wild-type; ▲, rsp5/npi cells. B, protein extracts were prepared at the same time points and analyzed by Western immunoblotting with an anti-HA antibody. The blots were reprobed with an anti-phosphoglycerol kinase (PGK) antibody to provide loading controls.

FIGURE 3.

Ubiquitylation of Jen1-GFP-6His in end3Δ and rsp5/npi1 mutant cells. Parental 27061b (WT), rsp5/npi1, and end3Δ cells were transformed with pJEN1-GFP-6HIS. The cells were induced for the production of Jen1-GFP-6His in galactose for 2 h (30 °C). A, microscopy images of Jen1-GFP-6His in living cells before and after the addition of 2% glucose for 30 min. B, protein extracts from induced parental and end3Δ cells were prepared before and at the indicated times after the addition of 2% glucose and were then analyzed by Western immunoblotting with an anti-GFP antibody. C, lysates of cells incubated for 10 min with glucose were fractionated, as described under “Experimental Procedures.” All experiments were conducted in identical conditions of growth and cell fractionation. Solubilized membranes were incubated with Ni²⁺-NTA beads. The bound fraction corresponding to His₆-tagged ubiquitylated proteins were eluted by buffer containing 200 mm imidazole. Aliquots of solubilized membranes and bound fractions were resolved by electrophoresis and analyzed by Western immunoblotting (WB) with anti-GFP and anti-Ub antibodies. Note that this particular anti-Ub antibody hardly recognizes mono- and diubiquitins. S, solubilized membrane fractions; B, purified His₆-tagged proteins. The sizes of molecular weight markers are indicated.

FIGURE 4.

Effect of Lys to Arg mutations on the stability of Jen1-GFP. W303-1A jen1Δ cells transformed with either pSP2 (JEN1-GFP), pSP3 (JEN1-K9R-GFP), or pSP4 (JEN1-K338R-GFP) were induced for the production of Jen1-GFP in lactic acid for 4 h at 30 °C. A, acid lactic uptake was measured at the times indicated after the addition of 2% glucose. The results are percentages of initial activities. ■, Jen1-GFP; ▿, Jen1-K9R-GFP; ▵, Jen1-K338R-GFP; B, protein extracts collected at the same time points were analyzed by Western immunoblotting with an anti-GFP antibody. The blots were reprobed with an anti-phosphoglycerol kinase (PGK) antibody to provide loading controls. C, microscopy images of Jen1-GFP in living cells at the time points indicated after the addition of 2% glucose. WT, wild type (Jen1-GFP).

FIGURE 5.

Ubiquitylation and stability of Jen1-GFP in SUB280 and SUB413 cells. SUB280 and SUB413 cells producing WT or Ub-K63R ubiquitin, respectively, were transformed with pJEN1-GFP-6HIS. Cells were induced for the production of Jen1-GFP-6His in galactose for 2 h at 30 °C. A, microscopy images of Jen1-GFP-6His in living cells at the time points indicated after the addition of 2% glucose. B, protein extracts collected at the indicated times were analyzed by Western immunoblotting with an anti-GFP antibody. C, lysates of 5-min glucose-incubated cells were fractionated, as described under “Experimental Procedures.” All experiments were conducted in identical conditions of growth and cell fractionation. Solubilized membranes were incubated with Ni²⁺-NTA beads. The bound fraction corresponding to His₆-tagged ubiquitylated proteins was eluted by 200 mm imidazole-containing buffer. Aliquots of solubilized membranes and bound fractions were resolved by electrophoresis and analyzed by Western immunoblotting with an anti-GFP antibody. S, solubilized membrane fractions; B, purified His₆-tagged proteins. D, SUB280 and SUB413 cells were transformed with pSP2 (JEN1-GFP). Cells were induced for the production of Jen1-GFP in lactic acid for 4 h at 30 °C before the addition of glucose for 5 min. Left, microscopy images of Jen1-GFP in living cells at the time points indicated after the addition of 2% glucose. Right, protein lysates collected at the indicated times were analyzed by Western immunoblotting with an anti-GFP antibody. The sizes of molecular weight markers are indicated.