SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy

Brian D. Slaughter, Joseph M. Huff, Winfried Wiegraebe, Joel W. Schwartz, Rong Li

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Sterile-alpha-motif (SAM) domains are common protein interaction motifs observed in organisms as diverse as yeast and human. They play a role in protein homo- and hetero-interactions in processes ranging from signal transduction to RNA binding. In addition, mutations in SAM domain and SAM-mediated oligomers have been linked to several diseases. To date, the observation of heterogeneous SAM-mediated oligomers in vivo has been elusive, which represents a common challenge in dissecting cellular biochemistry in live-cell systems. In this study, we report the oligomerization and binding stoichiometry of high-order, multi-component complexes of (SAM) domain proteins Ste11 and Ste50 in live yeast cells using fluorescence fluctuation methods. Fluorescence cross-correlation spectroscopy (FCCS) and 1-dimensional photon counting histogram (1dPCH) confirm the SAM-mediated interaction and oligomerization of Ste11 and Ste50. Two-dimensional PCH (2dPCH), with endogenously expressed proteins tagged with GFP or mCherry, uniquely indicates that Ste11 and Ste50 form a heterogeneous complex in the yeast cytosol comprised of a dimer of Ste11 and a monomer of Ste50. In addition, Ste50 also exists as a high order oligomer that does not interact with Ste11, and the size of this oligomer decreases in response to signals that activate the MAP kinase cascade. Surprisingly, a SAM domain mutant of Ste50 disrupted not only the Ste50 oligomers but also Ste11 dimerization. These results establish an in vivo model of Ste50 and Ste11 homo- and hetero- oligomerization and highlight the usefulness of 2dPCH for quantitative dissection of complex molecular interactions in genetic model organisms such as yeast.

Original languageEnglish (US)
Title of host publicationSpectroscopy
Subtitle of host publicationNew Uses and Implications
PublisherApple Academic Press
Pages203-229
Number of pages27
ISBN (Electronic)9781466562318
ISBN (Print)9781926692821
StatePublished - Apr 19 2016

ASJC Scopus subject areas

  • Physics and Astronomy(all)
  • Engineering(all)

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    Slaughter, B. D., Huff, J. M., Wiegraebe, W., Schwartz, J. W., & Li, R. (2016). SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy. In Spectroscopy: New Uses and Implications (pp. 203-229). Apple Academic Press.