Thus, the targeting mechanisms are likely dependent on cell shape and local bacterial envelope composition. Our recent analysis of the C. crescentus localisome has identified 3 proteins that localize specifically to the stalk (Fig. 1) (4). The limited number of proteins that localize to the stalk provides a convenient basis for initial studies on the general mechanisms of protein localization. I will use these 3 stalk proteins (CC0498, CC1953, and CC3022) to probe the mechanisms of stalk localization in C. crescentus.
Very little is known about these 3 stalk proteins. Only CC1953 is part of a predicted operon. This operon contains 23 genes including 9 hypothetical proteins and the 14 nuo genes encoding NADH hydrogenase I subunits. None of the stalk proteins have a predicted function, nor do they have orthologs in a wide range of bacterial species. The .
sequences of the stalk proteins suggest that they are either inner-membrane proteins (CC0498 and CC1953) or of unknown location (CC3022) (12). However, none of these proteins have a known signal sequence for secretion. Despite the lack of predicted structure or function of these proteins, the fact that xylose-inducible mCherry fusions of these proteins localize to the stalk makes these proteins useful tools as markers for probing mechanisms of localization.
The goal of Aim 1 is to identify the general mechanisms which regulate stalk localization. I will first determine whether proteins are targeted directly to the stalk, or whether they arrive by diffusion and are then captured. I will also investigate the role of the bacterial envelope and cell shape in protein localization.
A. Is delivery of proteins to the stalk the result of direct targeting or diffusion and capture?.
In Shigella flexneri the virulence protein IcsA (VirG) is localized to one cell pole (13). IcsA is delivered directly to one pole where it is then free to diffuse throughout the membrane (14).