| A fundamental problem in cell biology is the generation of structure within cells. One level of subcellular organization involves the separation of constituents into membrane-bound compartments which may be further divided into specialized domains. For instance, one contiguous membrane system comprises the rough endoplasmic reticulum (ER), the smooth ER, and the inner and outer membranes of the nuclear envelope (NE). Although the ER membrane is well characterized, the composition of the NE and how it is functionally separated from the ER is poorly understood. One goal of this thesis was to better understand the NE by identifying membrane proteins specific to it. We developed a general approach to identify proteins based on their subcellular localization in tissue culture cells. In this visual screen, small pools of a cDNA library encoding fusion proteins to GFP were expressed in cells, and positive pools were identified by fluorescence of the structure of interest, such as the nuclear rim. Candidate cDNA clones were then purified from positive pools by sib-selection.; By this approach we identified nurim, the fifth reported inner nuclear membrane protein. Structurally, nurim is unique among this group in that it does not contain a prominent nucleoplasmic domain responsible for targeting, and localization does not appear to be mediated by binding to chromatin or to laminA/C. Yet it interacts with nuclear components very tightly.; The cytoskeleton provides another level of subcellular organization. Using the visual screen, we have identified a novel protein, MIR1, which associates with the microtubule cytoskeleton. MIRI is highly expressed in neuronal cells, as well as in thymus and testis. In cell lines it is more prominently localized at centrosomes than with microtubules, suggesting the presence of mechanisms for specific targeting. In both cases, the association is cell cycle regulated, and can be mediated by cdk kinases.; Novel proteins can thus be obtained by a visual screen. Identification of proteins by their subcellular localization is a useful approach in situations where the structures are difficult to manipulate biochemically or where genetic screens are not practical. |
