Involvement of the exomer complex in the polarized transport of Ena1 required for Saccharomyces cerevisiae survival against toxic cations

Abstract

Exomer is an adaptor complex required for the direct transport of a selected number of cargoes from the trans-Golgi network (TGN) to the plasma membrane in Saccharomyces cerevisiae However, exomer mutants are highly sensitive to increased concentrations of alkali metal cations, a situation that remains unexplained by the lack of transport of any known cargoes. Here we identify several HAL genes that act as multicopy suppressors of this sensitivity and are connected to the reduced function of the sodium ATPase Ena1. Furthermore, we find that Ena1 is dependent on exomer function. Even though Ena1 can reach the plasma membrane independently of exomer, polarized delivery of Ena1 to the bud requires functional exomer. Moreover, exomer is required for full induction of Ena1 expression after cationic stress by facilitating the plasma membrane recruitment of the molecular machinery involved in Rim101 processing and activation of the RIM101 pathway in response to stress. Both the defective localization and the reduced levels of Ena1 contribute to the sensitivity of exomer mutants to alkali metal cations. Our work thus expands the spectrum of exomer-dependent proteins and provides a link to a more general role of exomer in TGN organization.

FIGURE 1:

Comparative sensitivity between mutant strains defective on exomer or different cation transporters. (A) Growth of different yeast strains on YEPD plates supplemented with increased concentrations of the indicated compounds. Gradient plates from 0 to the specified maximum concentration were made as described under Materials and Methods. (B) Sensitivity of the indicated strains to hygromycin or Li⁺ in the absence or presence of 0.1 M KCl. Note the full suppression of hygromycin sensitivity on KCl-containing plates compared with the limited effect on Li⁺ sensitivity.

FIGURE 2:

The localization of PM transporters in exomer mutants. (A) Localization of different PM transporters in wild-type and chs5∆ strains. Proteins were chromosomally appended with the GFP at their C-terminus, except Trk1-GFP, which was expressed from plasmid pRS414. All proteins were visualized in cells growing in nonbuffered SD media except Ena1-GFP, which was also visualized at pH 7.0. Note the similar localization in wild-type and chs5∆ strains. (B) Rubidium uptake in the different mutants and (C) intracellular levels of potassium. The strain labeled ∆∆∆∆ corresponds to strain YAS563-16a in which all four ChAPs have been deleted (see Table 1).

FIGURE 3:

Multicopy suppression analysis by HAL genes. (A) Growth of the wild-type and chs5∆ strains transformed with the indicated genes on SD media supplemented with the indicated compounds. (B) Drop assays of chs5∆ transformed with different HAL genes. Wild-type strain is used as the control. In all cases, HAL genes were overexpressed from multicopy plasmids (YEp351 or Yep352). Cells were grown in selective SD to ∼2 × 10⁷ cells/ml media and serially diluted before spotting onto YPD plates containing either 0.2 M LiCl, 0.2 M (NH₄)₂SO₄, or 1 M NaCl. Plates were incubated 2–3 d at 30°C. (C) Deletion of PPZ1 rescues the chs5∆ growth defect on LiCl and NaCl plates. Drop assay with different strains. Plates were incubated 2–3 d at 30°C.

FIGURE 4:

Effect of the overexpression of different ionic transporters on the chs5∆ phenotypes. (A) chs5∆ cells were transformed with the indicated genes expressed from multicopy plasmids, grown on selective SD media to 2 × 10⁷ cells/ml, serially diluted and spotted onto the plates as in Figure 3. Growth was scored after 2 d at 30°C. Note the reduced sensitivity of the chs5∆ mutant to LiCl and NaCl, but not to (NH₄)₂SO₄, after ENA1 overexpression, while high levels of QDR2 improved growth on the three media. (B) Li⁺ extrusion in the indicated strains. Cells were preloaded with Li⁺ and transferred to fresh media. Cells were collected at the indicated times, and the internal amounts of Li⁺ were measured by atomic absorption. Data are presented as the percentage of the original values of the strain at 0 time. The results are the average of three independent cultures. See Materials and Methods for details. Note the limited extrusion capacity of the chs5∆ mutant.

FIGURE 5:

Assessing the role of exomer on Ena1 function. (A) Localization of a chromosomally tagged version of Ena1-GFP in wild type and chs5∆. Cells were grown on selective SD media to logarithmic phase and then incubated under the indicated conditions. The percentage of cells showing polarized distribution of the fluorescence signal associated with Ena1-GFP is given as average values with standard deviations. (B) Polarization coefficients for any measured cell (n = number of cells) in the experiment described in A. See Materials and Methods for details. (C) Ena1-GFP levels before and after alkalinization of SD media under the same experimental conditions as in A. (D) Levels of Ena1-GFP expressed from the tetO promoter. Cells were grown in the presence of 10 mg/ml dox and transferred for 2 h to fresh media supplemented with the indicated concentrations of the drug. (E) Ena1-GFP was visualized by fluorescence microscopy after growth on the indicated dox concentration for 2 h. Cation treatment was performed for additional 30 min. (F) Levels of Ena1-GFP polarization in the same experiment. The results are the average of at least three independent experiments counting at least 50 cells in each experiment. Note the higher polarization of Ena1-GFP in wild type in all conditions tested. See also Supplemental Figure S3 for a complementary quantitative analysis on Ena1-GFP polarization.

FIGURE 6:

Localization of PM proteins in chs5∆ mutant cells. (A) Hydrophobic profiles of the indicated proteins showing predicted transmembrane domains (gray squares) and signal peptide (SP) sequences. The postranslational modifications that have been experimentally described for these proteins are also indicated: P (phosphorylation) and K (ubiquitination). The profiles are drawn to approximate scale. (B) Representative images showing protein localization in wild-type and chs5∆ cells. The dependence of these proteins on Sro7 that has been previously described (Forsmark et al., 2011) is indicated, as well as their predicted nature as Type I or II TM proteins. Chs3 and Pin2 have been described as bona fide exomer cargoes. Note how Ena1 polarization, but not PM arrival, is defective in the chs5∆ mutant. The absence of exomer did not affect the localization of the other proteins. See Figure 2A for additional images on other permeases.

FIGURE 7:

Defective RIM101 signaling in the exomer mutant. (A) Comparative phenotypes of rim101∆ and chs5∆ mutants. Note the similar phenotypes observed and the additive effect of both mutations. (B) Immunoblot of Rim101 proteolytic processing at the indicated pHs. Cells contain a modified version of Rim101 with an internal 3xHA tag. Note the absence of processing in the chs5∆ mutant compared with the control. (C) Visualization of processing spots using yeast cells containing chromosomally tagged versions of Rim13 and Rim20 proteins. Note the increasing numbers of spots for both proteins after alkalinization of the media for 1 h in wild type, which is absent in the chs5∆ mutant. Right panel shows the quantitative results for this experiment, which are the average of three independent experiments. (D) Levels of Ena1-GFP from its endogenous locus after expression of the constitutively processed Rim101^C from the pRS315 plasmid. Cells were grown O/N in selective SD media and refreshed in YEPD media for 2 h. (E) Drop assay of strains transformed with the constitutively expressed form of Rim101 (pRS315::RIM101^C) on the indicated media. Note the moderate improvement of growth promoted by Rim101^C in the chs5∆ mutant under all conditions tested. (F) Rim21-x2GFP localization in the indicated strains/conditions. Note the lower cortical localization of the protein in the chs5∆ mutant independently of the media pH. The quantitative results are the average of at least three independent experiments counting at least 120 cells in each experiment. (G) Phenotypes of the indicated mutants in different media. Note that the absence of Aps1 restores wild-type calcofluor sensitivity of the chs5∆ mutant but not its growth on Na⁺ or Li⁺ plates.

FIGURE 8:

Scheme for exomer’s role on Ena1 function. (A) Simplified scheme of the control of alkali metal cation transport across the PM in S. cerevisiae. (B) Exomer controls alkali metal cation sensitivity by directly controlling Ena1 polarization and indirectly controlling Ena1 levels through the activation of the RIM101 signaling pathway.