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USO1 is one of the essential genes in Saccharomyces cerevisiae whose gene products participate in protein transport from the endoplasmic reticulum to the Golgi apparatus. This product was purified to homogeneity. Electron microscopic study revealed that it has a single or double globular domain with a long tail and that the molecule is a dimer. A peak position of the distribution of rod length was 154.5 nm, in agreement with the secondary structure prediction that it has a long alpha-helix at the carboxyl terminus. Probability of coiled-coil formation was also predicted from the primary structure of the product, which asserts that it has a long alpha-helical coiled-coil at the carboxyl-terminal region with some interruptions. Certainly, the electron microscopic image of this molecule had some hinges within the rod region. The distance was measured between the globular domain and the hinges. Two peaks of the distribution of the hinge position exist at 23.1 and 85.5 nm from the globular domain. This is consistent with the predicted positions of interruption. These results give new experimental evidence that Uso1 protein is a dimer and has an alpha-helical coiled-coil tail with two globular heads.

Saccharomyces cerevisiae uso1-1 mutant stops the transport of secretory proteins from the endoplasmic reticulum to the Golgi apparatus at 37 degrees C. We found that this temperature-sensitive defect was suppressed either by increasing the concentration of calcium ion in the medium or by introducing in the cell the SLY genes which suppress the defect of Ypt1 protein, a small GTP-binding protein. The common phenotype and suppression of the mutants suggest that Uso1 and Ypt1 proteins function in the same process of protein transport, i.e., targeting or fusion of the transport vesicles to the Golgi membrane.

We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein.

We have previously shown that USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a hydrophilic protein of 1790 amino acids. The sequence of carboxyl-terminal 1010 amino acids was predicted to have an alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. Antibodies raised against partial sequences of the Uso1 polypeptide reacted with a 200 kDa protein in Western blots of the wild-type yeast proteins. The Uso1 protein was found predominantly in the soluble fraction and displayed a molecular mass of 800-900 kDa in gel filtration when globular protein were used as molecular mass standards. In sucrose density gradient centrifugation, however, the Uso1 protein cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa). These results suggest that, in its native state, the Uso1 protein forms a nonglobular oligomer.