PROJECT SUMMARY Most cellular functions are carried out by multisubunit protein complexes. Transcribe and translate as much as you want, but odds are if your protein doesn’t assemble properly with other proteins, the gene might as well be off. As the final step in expression of most genes, protein complex assembly is hugely understudied, and it has only recently become clear that the textbook model of diffusion-limited collisions between individual molecules is inadequate. Septin proteins are found as cytoskeletal filaments in many eukaryotic cells and participate in a wide variety of cellular functions. The building blocks of septin filaments are rod-shaped septin complexes composed of distinct septin subunits. Multiple septins “compete” to occupy the same position in, and confer specialized properties to, septin complexes, but it is not fully understood how specific subunits are “chosen” to assemble the functionally appropriate complexes. How individual septins occupy specific positions within complexes is one of the oldest questions in septin biology. Disease-causing septin mutations highlight the importance of answering this question. Our lab is among the leaders in this field and in the next five years we want to use the powerful tools we have developed in budding yeast to address specific knowledge gaps. For other cytoskeletal proteins, achieving the conformation competent for complex assembly requires help from molecular chaperones. How does chaperone-assisted de novo septin folding fit into the pathway of septin complex assembly? Our recent work established the step-wise pathway of septin hetero-octamer assembly, and identified septin-interacting chaperones that engage a septin-septin interaction interface and are necessary for efficient septin folding. We will use a combination of genetics, cell biology and biochemistry to determine how chaperone action sets the stage for septin-septin encounters during complex assembly. Recent studies show that assembly of many complexes occurs co-translationally, and we find chaperone requirements for efficient septin translation. To what extent is septin complex assembly co-translational? We will investigate septin- chaperone and septin-septin interactions in the context of active translation. We previously identified key residues in septin-septin interaction interfaces that mediate “partner recognition” during assembly and dictate the subunit composition within complexes. An enduring mystery is how the two subunits at the “ends” of yeast septin hetero-octamers always match. How do allosteric conformational changes across septin-septin interaction interfaces direct the specificity of septin complex subunit composition? We will determine the mechanistic basis of this phenomenon and determine the phenotypic consequences of inappropriate “mixing” of septin subunits within complexes. Finally, it is not known how, once made, a septin complex is remodeled to incorporate new subunits during cellular differen...