A proteomics analysis of yeast Mot1p protein-protein associations: insights into mechanism

Abstract

Yeast Mot1p, a member of the Snf2 ATPase family of proteins, is a transcriptional regulator that has the unusual ability to both repress and activate mRNA gene transcription. To identify interactions with other proteins that may assist Mot1p in its regulatory processes, Mot1p was purified from replicate yeast cell extracts, and Mot1p-associated proteins were identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. Using this approach we generated a catalog of Mot1p-interacting proteins. Mot1p interacts with a range of transcriptional co-regulators as well as proteins involved in chromatin remodeling. We propose that interaction with such a wide range of proteins may be one mechanism through which Mot1p subserves its roles as a transcriptional activator and repressor.

Fig. 1.

Yeast Mot1p exists in heterodisperse high molecular weight complexes. Yeast WCE protein (4 mg), prepared from DPY107 cells, was fractionated by fast protein LC on a Superose 6 HR 10/30 column in the absence (middle panel) or presence (lower panel) of ethidium bromide (EtBr). Purified rMot1p (50 μg; top panel) and molecular mass standards (thyroglobulin (669 kDa), ferritin (440 kDa), and Catalase (232 kDa)) were fractionated in parallel. The column void volume (V₀) was measured using plasmid pRS305 (molecular mass = 3.4 MDa). Fractions were collected, and aliquots of fractions and WCE were precipitated, separated by SDS-PAGE, and electroblotted to PVDF membranes, and Mot1p was detected with polyclonal anti-Mot1p IgG (rMot1p) or monoclonal anti-HA-HRP IgG (WCE).

Fig. 2.

MudPIT identification of previously characterized Mot1p-associated proteins. A, results of MudPIT analysis for Mot1p and TBP/Spt15p. Top, control (Group 1 (G1)) = average number of peptide counts over the 12 control reactions. Experimental (Exp) (Group 2 (G2)) = average number of peptide counts over the seven experimental reactions. Group 2/Group 1 = experimental average/control average. q value, expressed as a percentage, measures the significance of the difference between Group 1 and Group 2 as calculated by SAM. p value was calculated by Mann-Whitney U test. Bottom, also shown are peptide counts, color-coded (see legend, right), for each independent MudPIT run: the first 12 columns are control runs; the last seven columns experimental runs. B, authentication of MS-identified Mot1p protein-protein interactions by co-IP analysis. WCE from DPY107 cells, which express HA₃-Mot1p, was immunoprecipitated with bead-bound anti-HA IgG that had been blocked overnight with a 50-fold mole excess of either HA or FLAG peptide. The protein content of the resulting IPs was analyzed by immunoblotting with affinity-purified rabbit polyclonal anti-TBP antibody or anti-HA-HRP IgG in the case of Mot1p. C, results of MudPIT analysis for NC2 ordered by descending molecular weight. D, authentication of Mot1p-NC2 subunit interactions by co-IP. Ncb1p/2p in IPs was scored by immunoblotting with affinity-purified rabbit polyclonal anti-Ncb1p and anti-Ncb2p antibodies. E, SAGA subunits identified by MudPIT analysis. F, authentication of Mot1p-SAGA subunit interactions by co-IP. SAGA subunits in the IPs were identified by immunoblotting with the indicated affinity-purified rabbit polyclonal antibodies.

Fig. 3.

Association of Mot1p with chromatin-remodeling complexes. A, results of MudPIT analysis for RSC. B, authentication of Mot1p-RSC subunit interactions by co-IP performed as in Fig. 2B. C, INO80 subunits identified by MudPIT analysis. D, authentication of Mot1p-INO80 subunit interactions by co-IP. E, ISW1 and ISW2 complex subunits identified by MudPIT analysis. F, authentication of Mot1p-ISW subunit interactions by co-IP. G, NuA4, Sin3, and FACT subunits identified by MudPIT analysis. H, authentication of Mot1p protein-protein interactions by co-IP analysis. Exp, experimental; G1, Group 1; G2, Group 2.

Fig. 4.

Association of Mot1p with DNA-binding transfactors and the general coactivator, Mediator. A, DNA-binding transfactors identified by MudPIT analysis. B, authentication of Mot1p-Msn2p interaction by co-IP analysis. C, Mediator subunit identified by MudPIT analysis. D, authentication of Mot1p-Srb4p interaction by co-IP analysis. Exp, experimental; G1, Group 1; G2, Group 2.

Fig. 5.

Interaction of Mot1p with proteins involved in DNA packaging and maintenance. A, proteins involved in DNA replication, repair, and recombination identified by MudPIT analysis. B, authentication of Mot1p-Rfa3p-GFP interaction by co-IP analysis. Exp, experimental; G1, Group 1; G2, Group 2.

Fig. 6.

Interaction of Mot1p with other classes of proteins. A, proteins of unknown function identified by MudPIT. B, authentication of Mot1p-YGR130C-GFP interaction by co-IP analysis. C, other Mot1p-interacting proteins identified by MudPIT. D, authentication of Mot1p protein-protein interactions by co-IP analysis. Exp, experimental; G1, Group 1; G2, Group 2.

Fig. 7.

Mot1p participates in multiple, distinct functional complexes. A, schematic illustration of a hypothetical, Mot1p-nucleated megacomplex. In A, B, D, and F, the protein (complex) used as bait in the immunopurification experiment is indicated by a rectangle, associated proteins are indicated by ovals, red lines are used throughout to indicate protein-protein interactions that have been described previously either via genetics or high throughput MS analyses, and black lines demarcate novel interactions observed in our analyses. B, schematic of identified interactions between protein complexes revealed by MudPIT analysis of NuA4/3 (via Eaf6p). C, MudPIT results for NuA4/3 immunopurification. TS, total spectra; UP, unique peptides; N, non-redundant peptides normalized to the control immunopurification. Results for the bait complexes are shown first followed by those for the identified interacting complexes. D, schematic representation of protein complexes found to interact with Mediator. E, MudPIT results for Mediator (Med6p) immunopurification. F, schematic representation of Ino80-associated protein complexes. G, MudPIT results for Ino80 (Nhp10p) immunopurification.

Fig. 7.

Mot1p participates in multiple, distinct functional complexes. A, schematic illustration of a hypothetical, Mot1p-nucleated megacomplex. In A, B, D, and F, the protein (complex) used as bait in the immunopurification experiment is indicated by a rectangle, associated proteins are indicated by ovals, red lines are used throughout to indicate protein-protein interactions that have been described previously either via genetics or high throughput MS analyses, and black lines demarcate novel interactions observed in our analyses. B, schematic of identified interactions between protein complexes revealed by MudPIT analysis of NuA4/3 (via Eaf6p). C, MudPIT results for NuA4/3 immunopurification. TS, total spectra; UP, unique peptides; N, non-redundant peptides normalized to the control immunopurification. Results for the bait complexes are shown first followed by those for the identified interacting complexes. D, schematic representation of protein complexes found to interact with Mediator. E, MudPIT results for Mediator (Med6p) immunopurification. F, schematic representation of Ino80-associated protein complexes. G, MudPIT results for Ino80 (Nhp10p) immunopurification.

Fig. 8.

Models for the involvement of Mot1p-associated proteins in transcriptional activation and repression. A, direct recruitment of Mot1p by DNA-bound transactivator proteins. Either Mot1p (blue) or Mot1p complexed with TBP (orange) may be recruited to a target promoter by direct interaction with a DNA-bound transactivator (green; DBD, DNA-binding domain; AD, activation domain). Recruitment of a Mot1p-TBP heterodimer is a mechanism by which TBP could be delivered to a target promoter, thereby leading to PIC formation and transcription activation (92). B, indirect recruitment of Mot1p by enhancer-bound coactivators. Mot1p or Mot1p-TBP could be recruited by a coactivator protein or protein complex (tan) that has itself been recruited to the enhancer by interaction with a DNA-bound transactivator. Subsequently Mot1p-TBP recruitment could again result in delivery of TBP to the promoter, or Mot1p may act in concert with coactivators to modulate chromatin structure, thus allowing stable binding of TBP to the promoter, PIC formation, and activation of transcription. C, indirect recruitment of Mot1p by repressor element-bound corepressors. To effect repression of transcription, Mot1p could be recruited by a corepressor protein or protein complex (yellow) that is bound to repressor cis-elements via interaction with a DNA-bound transcription repressor (green/red; SD, silencing domain), leading to alterations in the post-translational modification status of chromatin and/or the removal of TBP from the targeted promoter.