Usol Protein Contains a Coiled-Coil Rod Region Essential for Protein Transport from the ER to the Golgi Apparatus in Saccharomyces cerevisiae1

Abstract

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.

Molecular Characterization of Golgin-245, a Novel Golgi Complex Protein Containing a Granin Signature

“…Large domains of coiled-coils have also been noted in other autoantigens including the nuclear mitotic antigen (NuMA) (27), lamin B (28), and myosin heavy chain (29), and one or two coiled-coils have been noted in the 52-kDa SS-A/Ro (30) and 80/86-kDa Ku antigens (31). This motif has also been described in a number of other prokaryotic and eukaryotic proteins, most notably Golgi complex proteins (32,33), the streptococcal M protein (29), products of the nuclear oncogenes c-fos and c-jun (34), viral fusogenic proteins (35), and the envelope glycoproteins of HIV1 and other viruses (36). Although certain proteins with coiled-coils are able to bind DNA and are thought to be predominantly regulatory in their function (37), this structure also mediates the dimerization of certain transactivators (38) and is essential for recombination of certain viral proteins (39) and oligomerization of viral envelope proteins (40).…”

Sequential Involvement of p115, SNAREs, and Rab Proteins in Intra-Golgi Protein Transport

“…This conditional mutant yeast strain expresses a truncated form of Uso1p, the yeast homologue of p115 (39,45). The mutant Uso1p lacks the vesicle-binding domain (40,76), and the thermosensitive phenotype can be suppressed by the overexpression of SNAREs (77), suggesting that SNAREs can bypass the function of Uso1p and mediate the docking of vesicles.…”

Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

“…A possible role for CC2-CC4-AD was suggested by the uso1-1 and uso1-11 yeast mutants that contain intact H1, H2 and CC1 ( Fig. 4A) but are functionally compromised (Seog et al, 1994), and the finding that deletion of the C-terminal region in bovine p115 inhibits exocytic traffic (Satoh and Warren, 2008). To identify functional domains within the C-terminal region of p115, we generated a deletion mutant p115/1-766 that is similar to the yeast uso1-11 (Fig.…”

“…Our studies were prompted by the functionally compromised uso1-1 and uso1-11 mutants (Seog et al, 1994;Yamakawa et al, 1996), and the finding that CC4 interacts with a subset of ER-Golgi SNAREs (Shorter et al, 2002). We generated three C-terminally truncated p115 mutants (p115/1-766, p115/1-820 and p115/1-934) and examined their effect on Golgi ribbon architecture.…”

“…4) are functionally compromised (Seog et al, 1994;Yamakawa et al, 1996). This suggests that domains CC2, CC3 and CC4, as well as the AD -all of which are missing in uso1-1 and uso1-11 -are important for the function of Uso1p and, perhaps, p115.…”