The overall objective of this proposal is to study the molecular mechanisms involved in the process of endocytosis. Our approach is to isolate and characterize animal cell mutants that are deficient in endocytic function. Moreover, we focus specifically on mutants that carry conditional-lethal defects. The agents that we use to isolate the mutants are highly potent protein toxins that require endocytic functions to express their lethal activities.
Our specific aims are three-fold: 1. Further studies on G.7.1, a Chinese hamster ovary cell mutant carrying a conditional-lethal defect in endocytic function: We have so far isolated two mutants that at elevated temperature (39.5 C) lose viability and in the process become resistant to protein toxins. We have studied one of these mutants, called G.7.1, in more detail and the results suggest that it contains a heat-sensitive lesion affecting vacuolar acidification. We propose to more precisely identify the lesion in this mutant and to use the mutant as a model to investigate what effect the lesion has on endocytic processes. 2. Isolation and characterization of more mutants: Our selection procedure is capable of rescuing many different kinds of mutants containing conditional-lethal defects in endocytic function. We propose to search for, and characterize, additional mutants. 3. Genetic characterization of mutants: With techniques of somatic cell genetics we will determine whether the lesions in our mutants are expressed dominantly or recessively. Mutants that contain recessive lesions will be classified into complementation groups. We also propose to isolate and characterize revertants of the mutants.
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