Our laboratory is interested in the formation and dissolution of both normal and pathological protein complexes in the cell with an emphasis on the role of molecular chaperones in this process. We have continued our studies on the role of Hsc70 in clathrin-mediated endocytosis by generating knockout mice of the HSC70 cochaperones, neuronal-specific auxilin and ubiquitously expressed GAK. These chaperones present clathrin to Hsc70 and therefore have a major role in uncoating clathrin from clathrin coated vesicles and chaperoning clathrin in the cytosol. To knock-out GAK, we had to make a conditional knock-out mouse since the conventional knock-out was embryonic lethal. Our GAK knockout studies showed that GAK was an essential gene during development and in adult mice. As for neuronal-specific auxilin, the newborn knockout mice had relatively high mortality and surviving pups generally had a lower body weight than the wild-type pups. The surviving mice had a normal life-span, but they probably survived only because GAK was up-regulated as much as 3-fold in the brains of both surviving neonate and adult auxilin knockout mice. Surprisingly, normal levels of GAK could not compensate for the absence of auxilin as shown by the observation that clathrin-mediated endocytosis was inhibited in the hippocampal neurons of the knockout mice. An increased number of clathrin-coated vesicles and empty cages were present at the knockout synapses both in situ and in primary neuronal cultures. These results show that auxilin has a specialized role in recycling synaptic vesicles in the brain, which can only be compensated for by marked up-regulation of GAK levels in mouse neurons Mouse embryonic fibroblasts (MEFs) were derived from this conditional GAK knockout mouse and GAK was disrupted in the MEFs by using adenovirus expressing Cre recombinase. Knocking-out GAK, completely blocked clathrin-mediated endocytosis in the MEFs. In addition, the clathrin organization of the MEFs was totally disrupted with loss of clathrin from both the plasma membrane and trans-Golgi network. To compensate for the loss of clathrin-mediated endocytosis, there was a marked increase in fluid phase uptake. Interestingly, in the absence of GAK, cell division was arrested, but the MEFs were still viable for several weeks. These results show that the absence of GAK and in turn the lack of chaperoning of clathrin in the cytosol by Hsc70 had a profound effect on multiple systems in the cell. In addition to our research on clathrin-mediated endocytosis, we also studied the propagation of prions, infective proteins that can misfold into an amyloid conformation both in yeast and mammalian cells. In yeast, the molecular chaperone, Hsp104, regulates the inheritance of several yeast prions including PSI+, which is the prion form of the translation termination factor Sup35p. By using live cell imaging, we found that Hsp104 not only severs prion seeds, but trims or reduces the size of the seeds. We found that when Hsp104 was inactivated by growing yeast in guanidine, the seeds became smaller because Hsp104 was still able to trim them. However, it could not sever them and the number of seeds decreases and prion curing occurred. However, when Hsp104 was completely inactivated by expressing a dominant negative Hsp104 mutant, Hsp104 no longer trimmed or severed the seeds and therefore the seeds both increased in size and decreased in number as curing occurred. Therefore, Hsp104 both trims the seed to maintain their steady-state size and severs them to maintain the steady-state number of seeds as the yeast divides.
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