The conserved ATPase Get3/Arr4 modulates the activity of membrane-associated proteins in Saccharomyces cerevisiae

Abstract

The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin-proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin-proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Deltaget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways.

Figure 1.—

get3 mutants suppress the temperature-sensitive lethality of npl4-1. (A) Diagram of the Get3 protein. The site of truncation in the get3^tn allele is shown (∨) along with the p-loop ATP-binding site (striped box). Predicted myristoylation sites are marked with asterisks. (B) get3 mutants rescue npl4-1 growth at 30°. Wild-type (WT), npl4-1, get3, and double-mutant strains were grown to log phase and then serially diluted and plated to rich media at 25° and 30° for 2 days.

Figure 2.—

get3 mutants suppress npl4-1 phenotypes. (A) Δget3 rescues ERAD-mediated CPY* degradation in npl4-1 cells. Wild-type (NPL4), npl4-1, and npl4-1 Δget3 mutant cells were grown to log phase and protein synthesis was halted by treatment with cyclohexamide. Samples were collected 30, 60, and 120 min after cyclohexamide treatment, separated by SDS–PAGE, and subjected to Western blot analysis with anti-CPY antibodies. (B) get3^tn restores OLE1 transcription in npl4-1 cells. Northern analysis was performed on total RNA isolated from wild type (WT), npl4-1, or npl4-1 with the suppressing mutations spt23^tn or get3^tn, using OLE1- and ACT1-specific DNA probes. Cells were either continuously grown at 25° or shifted to 37° for 2 hr (labeled as 25° and 37°, respectively) prior to RNA purification. The ratio of OLE1/ACT1 signal for each sample is given (at the bottom) relative to that for the WT 25° sample.

Figure 3.—

GET3 is coregulated with genes encoding components of the proteasome and the Cdc48-Npl4-Ufd1 complex. GET3 (thick box) is similarly expressed with many protein-degradation genes (boldface type) and genes encoding the Cdc48/Npl4/Ufd1 complex (thin boxes). The data set of expression profiling under various conditions from Eisen et al. (1998) was analyzed by hierarchical clustering using Average Link correlation (uncentered), and the GET3-containing cluster from analysis with TreeView software is shown. Treatments analyzed include cell cycle time courses, the diauxic shift, a time course during sporulation, and the altered expression of NDT80.

Figure 4.—

ER-membrane localization of Get3 requires Get2 and Get1. (A) Get3-EGFP localizes to the nuclear/ER membrane in rich media, by live cell fluorescence microscopy. Corresponding Nomarski image of cells is shown to the left. (B) Get3-EGFP cofractionates with both soluble and membrane-bound fractions. Cellular extract (CE) was separated into pellet and supernatant fractions following centrifugation at 13,000 rpm (P13 and S13, respectively). The S13 fraction was then subjected to ultracentrifugation at 100,000 rpm and separated into pellet and supernatant fractions (P100 and S100). Corresponding volumes from each isolated fraction were separated by SDS–PAGE and Western blotted with anti-GFP or anti-Sec62 antibodies as indicated. (C) The P13-associated fraction of Get3-EGFP is tightly membrane associated. The P13 fraction as in B was washed with either buffer alone (lanes 1 and 2) or buffer with 1 m NaCl (lanes 3 and 4), 0.2 m Na₂CO₃ pH 11 (lanes 5 and 6), 0.1% Triton X-100 (lanes 7 and 8), or 1% Triton X-100 (lanes 9 and 10). The samples were then recentrifuged and separated into pellet (P, odd lanes) or supernatant fractions (S, even lanes), which were analyzed by Western blotting as above. (D) Get3 biochemically copurifies with Get2 and Get1. Solubilized membranes from cells expressing Get3-TEV-proteinA (GET3-pA) or protein A alone (pA) were incubated with IgG Sepharose beads. After extensive washing, bound proteins were treated with TEV protease. Proteins released by this treatment were separated by SDS–PAGE and visualized by silver staining. Specific bands marked with a dot were excised for analysis by MALDI-TOF mass spectrometry and two of these bands were identified as Get2 and Get1 as labeled. The protein band corresponding to Get3 is also indicated. (E) Get3-EGFP mislocalizes in the absence of GET2 and/or GET1. Get3-EGFP was visualized by live-cell fluorescence microscopy in Δget1, Δget2, or Δget1 Δget2 cells grown in rich media to logarithmic phase. Images are representative of analysis of more than one clone for each genotype.

Figure 5.—

Δget3 rescues phenotypes of Δget2 and Δget1 cells. (A) Representative sporulated cells displaying wild-type or defective terminal sporulation phenotypes. Synchronously sporulated SK1 cells were fixed 36 hr after shift to sporulation medium (SPM) and nuclei were stained with DAPI. Nomarski (left) and DAPI (right) images representative of the indicated terminal (36 hr) sporulation phenotypes are shown. (B) Δget3 rescues terminal sporulation defects displayed by Δget2 cells. Synchronously sporulated wild-type (WT), Δget3, Δget2, and Δget3 Δget2 homozygous diploid yeast (SK1 background) were stained with DAPI, and cells falling into each of the four categories shown in A were counted. The average percentage of each phenotype is graphed with error bars depicting the standard deviation over three separate experiments. At least 200 cells were counted for each strain per experiment. (C) Δget3 suppresses the HU sensitivity of Δget2 and Δget1 cells. Wild-type (WT), Δget3, Δget1, Δget2, and double-mutant strains were grown to log phase and then serially diluted and plated to YPD or media containing 150 mm hydroxyurea at 25° for 2 days.