Takemitsu Mizunaga

Isolation and characterization of a yeast gene, MPD1, the overexpression of which suppresses inviability caused by protein disulfide isomerase depletion

MPD1, a yeast gene the overexpression of which suppresses the inviability caused by the loss of protein disulfide isomerase (PDI) was isolated and characterized. The MPD1 gene product retained a single disulfide isomerase active site sequence (APWCGHCK), an N‐terminal putative signal sequence, and a C‐terminal endoplasmic reticulum (ER) retention signal, and was a novel member of the PDI family. The gene product, identified in yeast extract, contained core size carbohydrates. MPD1 was not essential for growth, but overexpression of the gene suppressed the maturation defect of carboxypeptidase Y caused by PDI1 deletion, indicative of the related function to PDI in the yeast ER.

Striated and crystalline inclusions in the nuclei and cytoplasm of intact yeast cells and yeast protoplasts.

Inclusions arise in the cytoplasm and the nucleus when intact yeast cells or yeast protoplasts are incubated in Tris buffer with added KCl or NaCl, sorbitol or mannitol in the absence of utilizable nutrients. Three kinds of inclusions were seen; leaflets composed of parallel, frequently curved microtubules, banded rods, and crystals with hexagonal fine structure. The first kind was duly seen in the nucleus, the third more frequently in the cytoplasm, but rods were found in either of these situations. Inclusions were particularly numerous in yeast cells that had been cultured in a basal medium with low phosphorus content before they were exposed to Tris-KCl solutions. Nuclear and cytoplasmic inclusions were commonly found close to or in contact with membraneous structures. It is tentatively proposed that lack of nutrients may impair the maintenance of the organization of the cytoplasm of intact cells or protoplasts, and that under these conditions certain components of the cells may disintegrate. Some of the released constituents may then rearrange themselves into nuclear or cytoplasmic inclusions by a process of self-assembly.

Thyroid hormone binding protein contains glycosylation site binding protein activity

Several lines of evidence provided by other workers indicate that within the same species thyroid hormone binding protein, the beta-subunit of prolyl hydroxylase, and protein disulfide isomerase are the same protein. We sought to determine if glycosylation site binding protein, a lumenal protein of the endoplasmic reticulum, also has the same primary structure. To accomplish this the level of glycosylation site binding protein (GSBP) activity, measured by photolabeling with a glycosylation site peptide probe, was carried out in preparations of 3T3 cells and in E. coli transformed with human thyroid hormone binding protein cDNA. The results strongly support the idea that GSBP is identical to these other lumenal proteins of the endoplasmic reticulum.

The Role of Protein Disulfide Isomerase in Yeast

Most disulfide-containing proteins are extracellular and most extracellular proteins contain disulfide bonds. Secretory enzymes, polypeptide hormones, immunoglobulines, and other serum proteins all have disulfide bonds. These extracellular and cell surface proteins are translated on membrane-bound ribosomes and concurrently translocated into the lumen of the endoplasmic reticulum (ER) in a largely unfolded state. Several lines of evidence suggest that disulfide formation is an early event in the processing of such proteins, occurring on nascent chains in some cases, and usually completed on polypeptides located in the rough ER. Very little is known about the formation of these disulfide bonds compared on post-translational modifications such as signal-peptide cleavage and glycosylation.