Regulation of the Yeast Hxt6 Hexose Transporter by the Rod1 α-Arrestin, the Snf1 Protein Kinase, and the Bmh2 14-3-3 Protein

Abstract

Cell viability requires adaptation to changing environmental conditions. Ubiquitin-mediated endocytosis plays a crucial role in this process, because it provides a mechanism to remove transport proteins from the membrane. Arrestin-related trafficking proteins are important regulators of the endocytic pathway in yeast, facilitating selective ubiquitylation of target proteins by the E3 ubiquitin ligase, Rsp5. Specifically, Rod1 (Art4) has been reported to regulate the endocytosis of both the Hxt1, Hxt3, and Hxt6 glucose transporters and the Jen1 lactate transporter. Also, the AMP kinase homologue, Snf1, and 14-3-3 proteins have been shown to regulate Jen1 via Rod1. Here, we further characterized the role of Rod1, Snf1, and 14-3-3 in the signal transduction route involved in the endocytic regulation of the Hxt6 high affinity glucose transporter by showing that Snf1 interacts specifically with Rod1 and Rog3 (Art7), that the interaction between the Bmh2 and several arrestin-related trafficking proteins may be modulated by carbon source, and that both the 14-3-3 protein Bmh2 and the Snf1 regulatory domain interact with the arrestin-like domain containing the N-terminal half of Rod1 (amino acids 1-395). Finally, using both co-immunoprecipitation and bimolecular fluorescence complementation, we demonstrated the interaction of Rod1 with Hxt6 and showed that the localization of the Rod1-Hxt6 complex at the plasma membrane is affected by carbon source and is reduced upon overexpression of SNF1 and BMH2.

Keywords: 14-3-3 protein; AMP-activated kinase (AMPK); arrestin; membrane transport; trafficking; yeast.

FIGURE 1.

Mapping of the domains required for the interaction between Snf1 and Rod1. A, strains transformed with plasmids containing the full-length SNF1 gene, the kinase domain (amino acids 1–391, SNF1-KD) or the regulatory domain (amino acids 392–633, SNF1-RD) fused to the LexA DNA-binding domain and the full-length ROD1, the arrestin domain-containing N terminus (amino acids 1–395, ROD1-Nt) or the (L/P)PXY motif-containing C terminus (amino acids 396–837, ROD1-Ct) fused to the Gal4 activation domain were grown to saturation in selective medium, serially diluted, and spotted onto plates with the indicated composition. Growth was recorded after 48–72 h. Identical results were obtained for three independent transformants. B, correct expression of all proteins in whole cell extracts was confirmed by immunodetection using the indicated antibodies. Molecular weight markers are indicated on the left, and direct blue staining of the membrane is shown in the bottom panel as the loading control.

FIGURE 2.

Snf1 interacts with Rod1 and Rog3 but not with other ART family members. A, strains transformed with plasmids containing the full-length SNF1 gene fused to the LexA DNA-binding domain and nine ART family member genes fused to the Gal4 activation domain were grown to saturation in selective medium, serially diluted, and spotted onto plates with the indicated composition. Growth was recorded after 48–72 h. Identical results were obtained for three independent transformants. No growth was observed in empty vector controls expressing the ART proteins (not shown). B, correct expression of the fusion proteins was confirmed by Western blotting as described in Fig. 1B.

FIGURE 3.

The 14-3-3 protein Bmh2 physically interacts with several ART family members. A, strains transformed with plasmids containing the BMH2 gene fused to the LexA DNA-binding domain, and nine ART family member genes fused to the Gal4 activation domain were grown to saturation in selective medium, serially diluted, and spotted onto plates with the indicated composition. Growth was recorded after 48–72 h. Identical results were obtained for three independent transformants. B, correct expression of the fusion proteins was confirmed by Western blotting as described in Fig. 1B.

FIGURE 4.

Bmh2 interacts with the Rod1 N terminus and Snf1 kinase activity improves this interaction. A, strains transformed with the indicated plasmids described in Figs. 1 and 3 were analyzed as described. Identical results were observed for three independent transformants. B, correct expression of the fusion proteins was confirmed by Western blotting as described in Fig. 1B. C, the indicated strains were grown to mid-log phase in selective medium, collected by centrifugation, and processed for the determination of β-galactosidase activity, as described under “Experimental Procedures.” The data represent the average values of triplicate determinations for three independent transformants. The error bars represent the standard deviation. β-Galactosidase activity was undetectable in the controls containing empty plasmids. D, the strains transformed with the indicated plasmids were analyzed for protein-protein interactions as described in Fig. 1.

FIGURE 5.

Rod1 co-immunoprecipitates with Hxt6. The BY4741 strain was transformed with plasmids containing the indicated genes, grown in raffinose-containing medium, treated with glucose for 30 min (final concentration, 2%), and processed for co-immunoprecipitation as described under “Experimental Procedures.” The purified proteins were separated on SDS-PAGE gels, transferred to nitrocellulose membranes, and successively analyzed with the indicated antibodies (upper panels). The bottom panels show the correct expression of fusion proteins in the proteins extracts employed for the co-immunoprecipitation experiments. Molecular weight markers are indicated on the left. Similar results were observed in three independent experiments.

FIGURE 6.

Visualization of the Rod1-Hxt6 complex in vivo using bimolecular fluorescence complementation. The HXT6-VC::HIS3 strain was transformed with the ROD1-VN or the empty plasmid, and the wild type strain was co-transformed with the VC empty plasmid and the ROD1-VN, as indicated. The cells were grown to mid-log phase in raffinose medium. A, images of the BiFC fluorescence and the overlay with the transmitted light are shown. B and C, the HXT6-VC::HIS3 strain expressing ROD1-VN was co-transformed with the endocytic vesicle marker DsRed-FYVE (B) or the TGN marker DsRed-Sec7 (C). Strains were grown to mid-log phase in raffinose medium and transferred to raffinose (RAF) or glucose (GLU) medium without methionine for 30 min to induce the expression of Rod1. The cells were visualized by confocal microscopy as indicated under “Experimental Procedures.” D, the HXT6-VC::HIS3 strain expressing ROD1-VN was grown to mid-log phase in raffinose medium, incubated with 2 μg/ml FM4-64 for 60 min, washed, resuspended in glucose medium without methionine, and visualized by confocal microscopy 60 min later. Representative cells are shown. Identical results were obtained for at least two independent transformants and in at least two experiments. E, the plasma membrane fluorescence/internal fluorescence ratio was calculated from the data described in B and C (a total of 126 untreated and 88 treated cells were quantified). The horizontal midline represents the median, and the box depicts the upper and lower quartiles. The whiskers denote the maximal and minimal fluorescence intensities (p value < 0.001).

FIGURE 7.

Rod1 interacts with Hxt1 in endocytic vesicles upon 2-deoxyglucose treatment. The wild type strain was transformed with plasmids encoding Hxt1-VC, Rod1-VN, or the corresponding empty vector controls as indicated. The cells were grown to mid-log phase in glucose medium. A, images of the BiFC fluorescence and the overlay with transmitted light are shown. B and C, the Hxt1-VC/Rod1-VN strain was co-transformed with the endocytic vesicle marker DsRed-FYVE (B) or the TGN marker DsRed-Sec7 (C). Strains were grown in glucose or raffinose containing medium and, where indicated, shifted to raffinose medium or treated with 2-deoxyglucose (0.2%) for 30 min. Representative confocal images are shown. In the left set of panels in B and C, the far-left images show the overlay, and the middle and right panels show the BiFC and DsRed channels, respectively. Treatments are indicated below each set of panels. Similar results were observed in at least two independent transformants.

FIGURE 8.

Dynamic regulation of Rod1 and the Rod1-Hxt6 complex in response to changes in carbon source and signaling proteins. A, protein accumulation of Rod1, Hxt6, and Hxt1 in response to carbon source. Strains containing genomic insertions of GFP were grown as indicated and processed for protein extraction and immunodetection using the α-GFP antibody. B, the indicated genomically integrated BiFC strains, generated as described under “Experimental Procedures,” were grown in raffinose-containing medium (Raf) to the end of the log phase and analyzed by confocal microscopy to determine the presence and the subcellular localization of the Rod1-Hxt6 interaction by bimolecular fluorescence complementation. Representative images of samples 60 min after glucose (Glu) addition are shown. C, the strains described in B were grown in raffinose-containing MY medium and treated for the indicated times with glucose (final concentration, 2%). 30 individual cells were analyzed for each condition using the ImageJ software to determine the percentage of fluorescence intensity associated to the plasma membrane. The data were processed as described under “Experimental Procedures.” The graphs represent the number of cells containing the indicated percentage of BiFC signal at the plasma membrane. The differences between the WT versus SNF1 and the WT versus BMH2 distributions were statistically significant (p value < 0.001) at all three time points, as assessed by nonparametric, two-tailed Mann-Whitney U tests. Similar results were observed in two separate experiments.

FIGURE 9.

Overexpression of either SNF1 or BMH2 delays the glucose-induced degradation of Hxt6. The indicated plasmids were transformed into the HXT6-GFP::HIS3 strain. These strains were grown and treated as indicated and processed for protein extraction and immunodetection. The α-GFP antibody detects the Hxt6-GFP fusion, and the α-HA antibody detects the Snf1-HA and Bmh2-HA fusions. The Snf1-HA and Bmh2 fusions migrate at ∼80 and 45 kDa, respectively.

FIGURE 10.

Working model for Hxt6 regulation by the Rod1-Snf1–14-3-3 signaling pathway. The 14-3-3 protein Bmh2 and the regulatory domain of Snf1 associate with the arrestin-like domain of Rod1. Rod1 forms a complex with Hxt6 at the plasma membrane, which facilitates its degradation upon glucose addition. Increased levels of Snf1 or Bmh2 reduce this association and delay Hxt6 degradation, presumably by reducing the efficiency of Rsp5-dependent ubiquitylation of the permease. MVB, multivesicular body; EE, early endosome; Vac, vacuole; Arr, arrestin-like domain; KD, kinase domain; RD, regulatory domain.