SAM Domain-Based Protein Oligomerization Observed by Live-Cell Fluorescence Fluctuation Spectroscopy

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.

Quantitative Measurement of Brightness from Living Cells in the Presence of Photodepletion

“…Applying this method to the measurements of EGFP in S. cerevisiae eliminates the scatter seen in Figure 1A and yields the expected brightness for monomeric EGFP at all concentrations (Figure 5). The standard deviation of segmented brightness analysis is ∼10% and represents a significant improvement over the uncertainty of previously reported brightness data in yeast [1], [25]. This value is close to the standard deviation achieved in mammalian cells.…”

Dual Modes of Cdc42 Recycling Fine-Tune Polarized Morphogenesis

“…FCCS assesses protein complex formation by measuring co-diffusion, or cross-correlation, of red- and green-labeled species. We showed previously that cross-correlation between mCherry and GFP-tagged proteins expressed at their endogenous levels in live yeast cells is an effective method to quantitatively examine cytosolic interactions (Slaughter et al, 2008; Slaughter et al, 2007). Strong cross-correlation (20%) was indeed observed between GFP-Cdc42 and Rdi1-mCherry in the cytosol of polarized yeast cells, compared to that of the negative control of unlinked GFP and mCherry (~4%).…”

“…Fluorescence cross-correlation spectroscopy (Bacia et al, 2006) data was obtained in live yeast as previously described (Slaughter et al, 2008; Slaughter et al, 2007). Briefly, GFP-Cdc42 and Rdi1-mCherry was excited using the 488 nm and 561 nm laser lines, respectively, of a Zeiss Confocor 3.…”

Tandem SAM Domain Structure of Human Caskin1: A Presynaptic, Self-Assembling Scaffold for CASK

“…Moreover, it is hard to know if the buffers used in vitro are representative of actual physiological conditions. To determine if the Caskin1 SAM-SAM self-association can occur in living cells, fluorescence cross-correlation spectroscopy (FCCS) was employed as has been done for other SAM-containing proteins (Slaughter et al, 2008). For these experiments two mammalian expression constructs were created in which the residues from the start of the caskin1 CID through the SAM domains were fused to mCherry (mCherry-Caskin1wt) or cyan fluorescent protein (CFP-Caskin1wt).…”