My research focuses on identifying molecular mechanisms underlying the assembly of macromolecular complexes, with a focus on multisubunit complexes formed by septin proteins. All cellular processes require the function of multisubunit complexes, and while much attention has been given to solving the final structures of such assemblies, comparatively little is known about how individual subunits adopt oligomerization-competent conformations and find their partner subunits in the crowded, dynamic cellular milieu. Below is a summary of the past research from our group.
• We first used unbiased genetic screening to find evidence that guanine nucleotide binding by septin proteins plays an entirely structural role during filament assembly, which can be bypassed by specific mutations in a key septin-septin oligomerization interface. Considering that one of these mutations has been found to cause male infertility in humans, these findings have compelling clinical relevance.
|The structure of a homodimer of the human septin SEPT2 bound to the nonhydrolyzable GTP analog GppNHp (PDB 3FTQ) with the residues corresponding to those we found in temperature-sensitive yeast rendered as spheres. GppNHp is shown in orange. From Weems, et al GENETICS 2013.|
• We then investigated a phenomenon in which, when cells have a choice between a wild-type and a mutant version of a given septin, mutant septins unable to bind nucleotides are discriminated against during higher-order assembly, despite their ability to function normally under the conditions tested. We discovered that prolonged interaction between nascent mutant polypeptides and cytosolic chaperones results in a kinetic delay in the acquisition by the mutant septin of a conformation competent for oligomerization with other septins. Our findings challenged the dominant paradigm that interactions between chaperones and their clients always promote mutant protein function, and provided a new way of thinking about how chaperone interactions might influence the ability of mutant alleles of proteins to perturb the functions of the wild type.
|Model for chaperone-mediated quality control of higher-order septin assembly. Nascent septin polypeptides emerging from the ribosome encounter a number of cytosolic chaperones during subsequent de novo folding. Wild-type septins efficiently adopt quasi-native conformations, thereby burying hydrophobic residues and escaping chaperone-mediated sequestration. Heterodimerization with other septins—the first oligomerization step toward septin filament assembly—occurs concomitant with exit from the chamber of the cytosolic chaperonin CCT (also called TRiC). Mutant septins that inefficiently fold the G heterodimerization interface are slower to achieve a conformation allowing chaperone release. Interactions with the prefoldin complex (PFD), the Hsp40 chaperone Ydj1, and the disaggregase Hsp104 are particularly prolonged, leading to a delay in availability of the mutant septin for hetero-oligomerization. From Johnson, et al Mol Biol Cell 2015.|
• We went on to develop an entirely new technique for monitoring, for the first time in living cells, the step-wise pathway of assembly of protein complexes, and used it to show how nucleotide-induced conformational changes drive the progression of such an ordered pathway for yeast septins. Moreover, we generated and validated a new model for the role of nucleotide hydrolysis by septins during oligomerization, and found that slow GTPase activity by a key septin subunit drives the formation of distinct hetero-oligomers comprised of alternative subunits. Our “GTPase timer” model predicted that changes in the ratio of GTP to GDP in the cytosol would influence the subunit composition of septin complexes. Our experiments supported this model and further provided a functional context for why septin complex composition is tuned to the metabolic state of the cell.
|Model for the step-wise assembly of yeast septin hetero-octamers. From Weems & McMurray, eLife 2017.|
• We studied the functions of the gametogenesis-specific septin subunits in yeast and determined that they play important and unique roles in guiding the biogenesis of new membrane and cell wall. As septins also play important, but poorly understood, roles in human gametogenesis, these findings are also relevant to human septin function.
|Model for the defects in prospore membrane (PSM) biogenesis in septin-mutant yeast cells. SPB, spindle pole body; LEP, leading edge protein. From Heasley & McMurray, Molecular Biology of the Cell 2016.|
• Finally, we studied the molecular basis of action of a small molecule (forchlorfenuron, FCF) thought to act specifically on septin filament assembly in non-plant eukaryotes and therefore used by many researchers as a “septin drug”. We found clear evidence of off-target effects, using four different eukaryotic cell types.
Our studies challenge the idea that thermodynamics (i.e., the affinities between proteins randomly colliding in solution) is the main driving force of protein complex assembly, and favor largely unappreciated roles for the kinetics of protein folding and cooperativity between protein-protein interactions. We are now focusing our attention on the mechanisms by which septin proteins fold in living cells, and how the insights we gained from the study of mitotic yeast septins apply to septins in other developmental contexts, and to oligomers composed of non-septin proteins.
Weems A, McMurray M. The step-wise pathway of septin hetero-octamer assembly in budding yeast. eLife 6. pii: e23689 (2017) doi: 10.7554/eLife.23689
McMurray MA. Coupling de novo protein folding with subunit exchange into pre-formed oligomeric protein complexes: the 'heritable template' hypothesis. Biomolecular Concepts 7:271-81 (2016) doi: 10.1515/bmc-2016-0023
Heasley LR, McMurray MA. Small molecule perturbations of septins. Methods in Cell Biology 136:311-9 (2016) doi: 10.1016/bs.mcb.2016.03.013
McMurray MA. Assays for genetic dissection of septin filament assembly in yeast, from de novo folding through polymerization. Methods in Cell Biology 136:99-116 (2016) doi: 10.1016/bs.mcb.2016.03.012
Schaefer RM, Heasley LR, Odde DJ, McMurray MA. Kinetic partitioning during de novo septin filament assembly creates a critical G1 "window of opportunity" for mutant septin function. Cell Cycle 15:2441-53 (2016) doi: 10.1080/15384101.2016.1196304
Garcia G 3rd, Finnigan GC, Heasley LR, Sterling SM, Aggarwal A, Pearson CG, Nogales E, McMurray MA, Thorner J. Assembly, molecular organization, and membrane-binding properties of development-specific septins. Journal of Cell Biology 212:515-29 (2016). doi: 10.1083/jcb.201511029
Heasley LR, McMurray MA. Roles of septins in prospore membrane morphogenesis and spore wall assembly in Saccharomyces cerevisiae.Molecular Biology of the Cell 27:442-50 (2016) doi: 10.1091/mbc.E15-10-0721
Johnson CR, Weems AD, Brewer JM, Thorner J, McMurray MA. Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast. Molecular Biology of the Cell 26:1323-44 (2015). doi: 10.1091/mbc.E14-11-1531
Heasley LR, Garcia G 3rd, McMurray MA. Off-target effects of the septin drug forchlorfenuron on nonplant eukaryotes. Eukaryotic Cell 13:1411-20 (2014) doi: 10.1128/EC.00191-14
McMurray MA. Lean forward: Genetic analysis of temperature-sensitive mutants unfolds the secrets of oligomeric protein complex assembly. BioEssays 36:836-48 (2014)
Weems AD, Johnson CR, Argueso JL, McMurray MA. Higher-order septin assembly is driven by GTP-promoted conformational changes: evidence from unbiased mutational analysis in Saccharomyces cerevisiae. Genetics 196:711-27 (2014) doi: 10.1534/genetics.114.161182
de Val N, McMurray MA, Lam LH, Hsiung CC, Bertin A, Nogales E, Thorner J. Native cysteine residues are dispensable for the structure and function of all five yeast mitotic septins. Proteins 81:1964-79 (2013) doi: 10.1002/prot.24345
Bertin A, McMurray MA, Thorner J, Peters P, Zehr E, McDonald KL, Thai L, Pierson J, Nogales E. Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae. Molecular Biology of the Cell 23:423-32 (2012) doi: 10.1091/mbc.E11-10-0850
Garcia G III, Bertin A, Li Z, Song Y, McMurray MA, Thorner J, Nogales E. Subunit-dependent modulation of septin assembly: Budding yeast septin Shs1 promotes ring and gauze formation. Journal of Cell Biology 195:993-1004 (2011) doi: 10.1083/jcb.201107123
McMurray MA, Stefan CJ, Wemmer M, Odorizzi G, Emr SD, Thorner J. Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis. Biological Chemistry 392:699 (2011). doi: 10.1515/BC.2011.091
McMurray MA, Bertin A, Garcia III G, Lam L, Nogales EE and Thorner J. Septin filament formation is essential in budding yeast. Developmental Cell 20:540-9 (2011) doi: 10.1016/j.devcel.2011.02.004
Bertin A, McMurray MA, Thai L, Garcia III G, Votin V, Grob P, Allyn T, Thorner J and Nogales EE. Phosphatidylinositol-4,5-bisphosphate promotes budding yeast septin filament assembly and organization. Journal of Molecular Biology 404:711 (2010) doi: 10.1016/j.jmb.2010.10.002
Garrenton LS, Stefan C, McMurray MA, Emr SD and Thorner J. Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling. Proceedings of the National Academy of Sciences of the United States of America 107:11805 (2010) doi: 10.1073/pnas.1005817107
McMurray MA and Thorner J. Septins: molecular partitioning and the generation of cellular asymmetry. Cell Division 4:18. (2009) doi: 10.1186/1747-1028-4-18
McMurray MA and Thorner J. Reuse, replace, recycle: Specificity in subunit inheritance and assembly of higher-order septin structures during mitotic and meiotic division in budding yeast. Cell Cycle8(2):195-203. (2009)
McMurray MA and Thorner J. Biochemical properties and supramolecular architecture of septin hetero-oligomers and septin filaments. In: Hall PA, Russell SEG, Pringle JR, eds. The Septins. Chicester, West Sussex, UK: John Wiley & Sons, Ltd., pp. 49-100. (2008)
McMurray MA Thorner J. Septin stability and recycling during dynamic structural transitions in cell division and development. Current Biology 18:1203 (2008) doi: 10.1016/j.cub.2008.07.020
Bertin A*, McMurray MA*, Grob P*, Park SS, Garcia G 3rd, Patanwala I, Ng HL, Alber T, Thorner J, Nogales E. Saccharomyces cerevisiae septins: supramolecular organization of heterooligomers and the mechanism of filament assembly. Proceedings of the National Academy of Sciences of the United States of America 105:8274 (2008) doi: 10.1073/pnas.0803330105 *these authors contributed equally to this work.
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