This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formins are conserved actin filament-nucleating factors, and are required specifically in yeast to assemble polarized arrays of actin cables. It is not yet understood how these formins function within the context of the whole cell, or how they are integrated into the various signaling pathways that regulate cytoskeletal organization in yeast. A number of formin-interacting proteins have been implicated, but many of these binding partners were identified using recombinant systems, such as two-hybrid assays or bacterially-expressed proteins. To identify binding partners for the endogenous formins, we intend to isolate a yeast formin through tandem-affinity purification, and subject the recovered samples to mass spectroscopy. To that end, we have COOH-terminally tagged the formin-encoding BNI1 gene. Based on genetic interactions and cytoskeletal and cell morphology, the protein is functional. Preliminary purifications underway have yielded samples highly enriched for Bni1p. With subsequent analysis of these, the nature of the complexes recovered can be probed through purifications from yeast bearing mutations of complex members, as well as yeast subjected to varying conditions that perturb the various signalling pathways that modulate cytoskeletal polarity in yeast.
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