Identification of lysines within membrane-anchored Mga2p120 that are targets of Rsp5p ubiquitination and mediate mobilization of tethered Mga2p90

Abstract

Mga2p90 is an endoplasmic reticulum (ER)-localized transcription factor that is released from the ER membrane by a unique ubiquitin (Ub)-dependent mechanism. Mga2p90 mobilization requires polyubiquitination of its associating membrane-bound Mga2p120 anchor and subsequent Mga2p120-Mga2p90 complex disassembly that is mediated by ATPase Cdc48p and its heteromeric Ub-binding adaptor Npl4p-Ufd1p. Although previous studies have identified the Ub ligase (i.e., Rsp5p) and ligase-binding site on Mga2p120 that play a role in this process, the amino acids of Mga2p120 that are targets of ubiquitination and promote Mga2p90 mobilization are unknown. We have identified, using mass spectrometry analysis of in vitro ubiquitinated Mga2p120-Mga2p90 complex, that lysine residues 983 and 985 contained within the carboxy-terminal domain of Mga2p120 are Rsp5p-directed Ub-conjugation sites. Mutation of these residues as well as proximally located lysine 980 results in suppression of Mga2p120 ubiquitination in vitro and in vivo, inefficient liberation of Mga2p90 by Cdc48p(Npl4p/Ufd1p)in vitro, and ER retention of Mga2p in cells. Moreover, mga2Delta/spt23ts harboring Rsp5p binding and conjugation mga2 mutants express low OLE1 (an Mga2p90 target gene) transcripts and display reduced growth. We conclude that residues 980, 983, and 985 are targets of Rsp5p-induced polyubiquitination and mediate Cdc48p(Npl4p/Ufd1p)-dependent Mga2p90-Mga2p120 separation and Mga2p90 mobilization.

Figure 1. Mga2p120 is ubiquitinated on lysine 983 and 985 by Rsp5p

(A) Microsomes from cells expressing FLAG-tagged Mga2p120-Mga2p90 complex were collected and the proteins from the microsomes were eluted and subjected to immunoprecipitation with anti-FLAG antibodies. In vitro ubiquitination assays were performed using the immunoprecipitated complexes and recombinant E1, E2 and E3 (Rsp5p) as described in supplemental information. Proteins were separated by SDS-PAGE and the gel was stained with coomassie blue. (B) Model depicting Mga2p120 (unprocessed membrane-bound form) and Mga2p90 (processed membrane-tethered form) with noted IPT domain, 2 ankyrin (Anks) repeats, and transmembrane domain. Also shown is the Rsp5p interaction motif LPKY, the ubiquitinated lysine residues that were identified (983 and 985) (blue) by MS/MS analysis (described in detail in supplemental information), and the lysine residue in proximity to the identified residues (980) (green) that was not covered by the MS/MS analysis.

Figure 2. Mutations at lysine 980, 983 and 985 of Mga2p120 abolish Rsp5p mediated poly-ubiquitination

(A) Mga2p120-Mga2p90 complexes were immunopurified from cells harboring galactose inducible ^FLAGMGA2, ^FLAGmga2Δlpky, ^FLAGmga2-2k or ^FLAGMga2-3k as described in supplemental information. The immunoprecipitated proteins were subjected to an in vitro ubiquitination assay using recombinant Rsp5p. Modified proteins were resolved by SDS-PAGE, transferred to a nitrocellulose membrane and western blotting was performed with anti-Ubiquitin and anti-FLAG antibodies. (B) Lysates from cells harboring the indicated expression constructs were subjected to immunoprecipitation with anti-FLAG antibodies. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and proteins were detected by western blotting with anti-Ubiquitin and anti-FLAG antibodies. (C) Immunopurified Mga2p120-Mga2p90 complexes from cells harboring ^FLAGMGA2, ^FLAGmga2Δlpky, ^FLAGmga2-2k or ^FLAGMga2-3k were liberated from the beads with FLAG peptide and used in in vitro pull down assay with immobilized ^GSTRsp5p. Bound material was resolved by SDS-PAGE, transferred to a nitrocellulose membrane and detected by western blotting with anti-FLAG antibodies. The amount of GST-Rsp5p used in the pull down assays was determined by Ponceau S staining of the blot prior to transfer. The left panel shows the input for each Mga2p protein and * denotes an alternatively processed Mga2p product. (D) Cells expressing HA-tagged Rsp5p and/or FLAG-tagged Mga2p proteins were lysed and immunoprecipitations were performed with anti-FLAG antibody. Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes and the membranes were probed with either anti-HA or anti-FLAG antibodies. The left panel shows input levels of Mga2p and Rsp5p proteins in the extracts as determined by western blotting. These and all other experiments presented here were repeated three times and similar results were obtained.

Figure 3. Lysine 980, 983 and 985 of Mga2p120 mediate mobilization of Mga2p90 from membranes in vitro and in vivo

(A) (left panel) Recombinant Cdc48p and Cdc48p^Npl4p/Ufd1p complex was prepared, resolved by SDS-PAGE gel and visualized by coomassie blue staining to assess purity as described in supplemental information. (right panel) Microsomes were isolated from cells containing ^FLAGMGA2, ^FLAGmga2pΔlpky, or ^FLAGmga2p-3k, and an in vitro mobilization assays were performed as described previously (12) with BSA, Cdc48p or Cdc48p^Npl4p/Ufd1p complex with or without Apyrase. Following completion of the assay the soluble and insoluble fractions were separated by ultracentrifugation and amounts of Mga2p products in the two fractions were determined by western blotting with anti-FLAG antibodies. (B) Cells harboring ^FLAGMGA2, ^FLAGmga2pΔlpky, or ^FLAGmga2p-3k were fixed and localization of Mga2p in these cells was determined by indirect immunofluorescence as described in supplemental information using anti-FLAG mouse monoclonal antibody. Cells were also co-stained with rabbit anti-Kar2p polyclonal antibodies. FLAG tagged proteins were visualized by an Alexa fluor 647 (red)-conjugated anti-mouse secondary antibody while Kar2p was visualized by FITC (green)-conjugated anti-rabbit secondary antibody. N=nuclear.

Figure 4. Lysine 980, 983 and 985 of Mga2p120 negatively affect its function in vivo

(A) mga2Δspt23-ts cells were transformed with plasmids harboring MGA2, mga2-k980r, mga2-k983r or mga2-k985r, mga2Δlpky and mga2-3k. Cells were grown up at 25°C and then switch to 34°C for 16 hrs. Cells were harvested, RNA was prepared and northern blotting was carried-out with radiolabeled OLE1. RNA loading was assessed by ethidium bromide staining of gel prior to transfer and levels of 28s and 18s rRNA are depicted in the bottom panel. (B) Cells transformed with the indicated constructs were grown-up at 25°C, serially diluted and plated on synthetic dropout media with or without oleic acid. Plates were incubated at 34°C for 2–3 days.