Studies on the intracellular protein transport in eukaryotic cells.

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Formerly we isolated a temperature-sensitive secretion mutant, uso1, in which protein transport from the ER to the Golgi apparatus is blocked at 37ﾟC.The uso1 gene was cloned and sequenced. The deduced amino acid sequence of Uso1 protein suggests that the protein contains 1790 amino acid residues and a C-terminal coiled-coil portion composed of 1100 amino acids. In this study we purified Uso1 protein from the lysate of Saccharomyces cerevisiae and observed the physico-chemical properties characteristic of a fibrous protein. Recently we succeeded to see directly the shape of the protein under the electron microscope (cooperative work with Prof.T.Wakabayashi of Univ.Tokyo). The Uso1 protein looks like myosin heavy chain composed of two heads and a long rod region of 150 nm just coincided with the predicted coiled-coil portion. As to the function of Uso1 protein it will not be a motor protein because we cannot find ATP binding sequence in the predicted head portion. Uso1 protein is suggested to participate in the membrane fusion step of secretory vesicles to the Golgi membrane from the study of suppressor mutation.In our previous work, we revealed that brefeldin A (BFA) specifically blocks intracellular protein transport between the ER and the Golgi apparatus in the yeast Candida albicans. Later we found that Schizosaccharomyces pombe became BFA-sensitive in the presence of 0.004% SDS.In the host-vector system of S.pombe, we cloned several DNA fragments which endowed the host BFA-resistance at the multi-copy state. We sequenced one DNA fragment and bfr1^+ (encoding 1530 amino acids) was found to be responsible for BFA-resistance. The gene has some similarity to the mammalian multi-drug resistance gene mdr. We also screened inhibitors of intracellular protein transport by a BHK cell-NDV virus system. Folimycin and SS33410 were selected as effective inhibitors and their targert molecule was found to be V-type H^+-ATPase.