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

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Sterile-alpha-motif (SAM) domains are common protein interactions motifs observed in organisms as diverse at 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 flourescence fluctuation methods. Flourescence 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), which endogenously expressed proteins tagged with GFP of mCherry, uniquely indicated that Ste11 and Ste50 form a heterogenous 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 decreased 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 and 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)
Article numbere1931
JournalPloS one
Issue number4
StatePublished - Apr 23 2008

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General

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