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

Slaughter BD, Huff JM, Wiegraebe W, Schwartz JW, Li R - PLoS ONE (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

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.

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1dPCH analysis of Ste50-GFP and Ste11-GFP probes homo-oligomerization.A. Example curves for GFP and mCherry (mCH.) controls in live yeast. B. Notched box plots of PCH fits, ranging from 18 to 30 individual, 7 second data traces from 5 to 10 cells. For auto-fluorescence measurements, data represents 7 measurements for mCherry and 15 measurements for GFP. 50 µs bins were used to generate the PCH distributions. C. Notched box plots of 1dPCH fits of GFP tagged species, with lines (same color scheme as in B) representing average brightness values of monomer, dimer, and trimer controls for a basis of comparison.
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pone-0001931-g003: 1dPCH analysis of Ste50-GFP and Ste11-GFP probes homo-oligomerization.A. Example curves for GFP and mCherry (mCH.) controls in live yeast. B. Notched box plots of PCH fits, ranging from 18 to 30 individual, 7 second data traces from 5 to 10 cells. For auto-fluorescence measurements, data represents 7 measurements for mCherry and 15 measurements for GFP. 50 µs bins were used to generate the PCH distributions. C. Notched box plots of 1dPCH fits of GFP tagged species, with lines (same color scheme as in B) representing average brightness values of monomer, dimer, and trimer controls for a basis of comparison.

Mentions: In vitro, the Ste11 and Ste50 SAM domains have been shown to mediate homo-oligomerization, but this has not been demonstrated in vivo when proteins are expressed at the endogenous levels. PCH analyzes the probability distribution of detected photons from a small confocal volume to calculate particle concentration and brightness, usually reported as the average number of molecules in the focal volume (N) and counts per second per molecule (CPSM), respectively (Figure 1C) [29], [40]. As a comparison technique, molecular brightness reflects the oligomeric state of the fluorescent species when compared to the brightness of a standard, for example, a known monomer or dimer of the same fluorescent molecule. For controls, yeast strains expressing monomeric, dimeric, and trimeric cytosolic GFP under the control of the BZZ1 promoter were constructed, as previously described (Table 1) [27]. The distribution of brightness values for 1dPCH measurements in live yeast cells were recorded using the Zeiss confocor 3 with 488 nm excitation and BP 505–540 nm emission collection (Materials and Methods). Box plots, as well as example curves, are shown in Figure 3A and 3B. The brightness distributions of these GFP species were easily distinguishable, providing the basis of comparison for oligomeric status of mobile GFP-tagged proteins in yeast. In addition, the BAT2 locus was replaced by cytosolic mCherry and the BZZ1 locus was replaced by mCherry-mCherry (Table 1). At the low excitation powers that are necessary to minimize photobleaching in our experiments, the brightness of mCherry was less than that of eGFP (Figure 3B) but, at over 2000 CPSM, still presented an improvement from other monomeric, red autofluorescent protein options [35], as expected based on improvements in photostability, and quantum yield [37], [38].


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

Slaughter BD, Huff JM, Wiegraebe W, Schwartz JW, Li R - PLoS ONE (2008)

1dPCH analysis of Ste50-GFP and Ste11-GFP probes homo-oligomerization.A. Example curves for GFP and mCherry (mCH.) controls in live yeast. B. Notched box plots of PCH fits, ranging from 18 to 30 individual, 7 second data traces from 5 to 10 cells. For auto-fluorescence measurements, data represents 7 measurements for mCherry and 15 measurements for GFP. 50 µs bins were used to generate the PCH distributions. C. Notched box plots of 1dPCH fits of GFP tagged species, with lines (same color scheme as in B) representing average brightness values of monomer, dimer, and trimer controls for a basis of comparison.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2291563&req=5

pone-0001931-g003: 1dPCH analysis of Ste50-GFP and Ste11-GFP probes homo-oligomerization.A. Example curves for GFP and mCherry (mCH.) controls in live yeast. B. Notched box plots of PCH fits, ranging from 18 to 30 individual, 7 second data traces from 5 to 10 cells. For auto-fluorescence measurements, data represents 7 measurements for mCherry and 15 measurements for GFP. 50 µs bins were used to generate the PCH distributions. C. Notched box plots of 1dPCH fits of GFP tagged species, with lines (same color scheme as in B) representing average brightness values of monomer, dimer, and trimer controls for a basis of comparison.
Mentions: In vitro, the Ste11 and Ste50 SAM domains have been shown to mediate homo-oligomerization, but this has not been demonstrated in vivo when proteins are expressed at the endogenous levels. PCH analyzes the probability distribution of detected photons from a small confocal volume to calculate particle concentration and brightness, usually reported as the average number of molecules in the focal volume (N) and counts per second per molecule (CPSM), respectively (Figure 1C) [29], [40]. As a comparison technique, molecular brightness reflects the oligomeric state of the fluorescent species when compared to the brightness of a standard, for example, a known monomer or dimer of the same fluorescent molecule. For controls, yeast strains expressing monomeric, dimeric, and trimeric cytosolic GFP under the control of the BZZ1 promoter were constructed, as previously described (Table 1) [27]. The distribution of brightness values for 1dPCH measurements in live yeast cells were recorded using the Zeiss confocor 3 with 488 nm excitation and BP 505–540 nm emission collection (Materials and Methods). Box plots, as well as example curves, are shown in Figure 3A and 3B. The brightness distributions of these GFP species were easily distinguishable, providing the basis of comparison for oligomeric status of mobile GFP-tagged proteins in yeast. In addition, the BAT2 locus was replaced by cytosolic mCherry and the BZZ1 locus was replaced by mCherry-mCherry (Table 1). At the low excitation powers that are necessary to minimize photobleaching in our experiments, the brightness of mCherry was less than that of eGFP (Figure 3B) but, at over 2000 CPSM, still presented an improvement from other monomeric, red autofluorescent protein options [35], as expected based on improvements in photostability, and quantum yield [37], [38].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.

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.

Show MeSH
Related in: MedlinePlus