QB3 Postdoc Seminar
Seminar | April 19 | 4:30-5:30 p.m. | 177 Stanley Hall
Adam Perez | Thorner Lab
Assembly of a septin-associated protein complex in Saccharomyces cerevisiae
During progression through each division cycle, Saccharomyces cerevisiae cells erect septin-based cytoskeletal structures with stereotypical states of organization at specific sub-cellular sites. Each higher-order septin ensemble serves a role in coordinating processes required for normal cell morphogenesis, including bud emergence, proper bud shape, and timely execution of cytokinesis. The septin arrays serve as scaffolds for highly localized recruitment of other proteins, enzymes and even lipids needed to promote the reactions necessary for passage through a specific cell cycle stage. Importantly, septin structures are central for establishment of cell polarity via an intricate interplay with the small Rho family GTPase Cdc42. After a cell has budded, septins assemble into filaments that form a collar at the neck between the mother cell and its daughter. A set of interacting proteins that localize at or near the septin collar, including Aim44, Nba1, Nap1, and Nis1, has been implicated in preventing Cdc42-dependent bud site re-establishment at the incipient site of cytokinesis. We are currently investigating the molecular mechanisms that direct these proteins to the septin collar in order to better understand how they control the axis of polarity over the course of the cell cycle. Using live cell imaging, we have observed that Aim44 co-localizes with septins from G1 phase through cytokinesis, whereas Nis1 and Nba1 only arrive at the bud neck during G2/M and continue to mark the cytokinetic site after cell septation. Nap1 localizes transiently to the bud neck during S phase. We have also found that expression levels play a critical role in targeting Aim44 and Nis1 to the bud-neck. When overproduced, both proteins shift their subcellular distribution predominantly to the nucleus (Aim44 localizes with the inner nuclear envelope, as well as at the plasma membrane, whereas Nis1 accumulates within the nucleus), indicating that these proteins normally undergo nucleocytoplasmic shuttling. our results indicate that a previously unappreciated level at which localization of septin-associated proteins is controlled is via regulation of their nucleocytoplasmic shuttling, which places constraints on their availability for complex formation with other partners at the bud neck. We are currently performing in vitro binding assays to determine the order of assembly of the Aim44/Nba1/Nis1/Nap1 complexes on septin filaments. It is our aim to utilize these biochemical observations to inform our understanding of how the assembly of these septin-associated protein complexes regulates localized Cdc42 activity within the cell.
Madeleine Jensen | Marqusee lab
Quantitative assessment of ribosome nascent chain energy landscapes
In the cell, the first time a protein has the opportunity to fold is co-translationally, and the ribosome presents a unique environment for protein folding. Attachment to the P-site tRNA, interactions with the ribosome surface, and the large mass of the ribosome may all modulate nascent chain folding and stability. Co-translational folding has been shown to improve protein folding efficiency compared to refolding in an idealized solution, but quantitative assessment of nascent chain stability and kinetics has been a longstanding challenge. Traditionally, these parameters could be determined by spectroscopic methods; however, the ribosome contains over 50 proteins and would obscure the signal from the nascent chain. Here, we use a simple, gel-based method pulse proteolysis to quantify nascent chain stability and kinetics while stalled on the ribosome. We show that the ribosome destabilizes the nascent chains studied and that this is likely due to an increase in the unfolding rate for the protein, rather than an effect on the folding rate. This suggests a chaperoning role for the ribosome that could help to limit the amount of time a nascent chain spends in misfolded conformations.