Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

ERp29 is a ubiquitous resident of the endoplasmic reticulum with a distinct role in secretory protein production.

ERp29 was recently characterized biochemically as a novel protein that resides in mammalian endoplasmic reticulum (ER). Here we applied immunochemical procedures at the cellular level to investigate the hypothesized role of ERp29 in secretory protein production. ERp29 was localized exclusively to the ER/nuclear envelope of MDCK cells using confocal immunocytochemistry and comparative markers of the ER lumen, ER/Golgi membrane, nuclei, and mitochondria. A predominant association with rough ER was revealed by sucrose-gradient analysis of rat liver microsomes. Immunohistochemistry showed ERp29 expression in 35 functionally distinct cell types of rat, establishing ERp29 as a general ER marker. The ERp29 expression profile largely paralleled that of protein disulfide isomerase (PDI), the closest relative of ERp29, consistent with a role in secretory protein production. However strikingly different ERp29/PDI ratios were observed in various cell types, suggesting independent regulation and functional roles. Together, these findings associate ERp29 primarily with the early stages of secretory protein production and implicate ERp29 in a distinct functional role that is utilized in most cells. Our identification of several ERp29-enriched cell types suggests a potential selectivity of ERp29 for non-collagenous substrates and provides a physiological foundation for future investigations.     

Overexpression of ERp29 in the thyrocytes of FRTL-5 cells

It was previously reported that the up-regulation of ERp29 mRNA depends on the levels of thyroid stimulating hormone (TSH) in the thyrocytes of FRTL-5 cells. In order to investigate the putative new function of ERp29 as an endoplasmic molecular (ER) chaperone, an ERp29-overexpressing FRTL-5 cell line was established. This cell line had approximately three times the levels of ERp29 protein and an enhanced level of thyroglobulin (Tg) secretion. The results showed both enhanced ERp29 expression and an interaction with the other ER chaperones such as GRP94, BiP, ERp72 and calnexin. In addition, ERp29 enhanced the expression of PKR-like ER kinase (PERK), which is a transmembrane protein located in the ER membrane. These findings suggest that ERp29 assists in protein folding as well as in the secretion of the secretory/plasma membrane proteins under close co-operation with other ER chaperones and the ER stress signaler, PERK.     

ERp29 is an essential endoplasmic reticulum factor regulating secretion of thyroglobulin

ERp29 is a ubiquitously expressed endoplasmic reticulum (ER) protein, which is found in the folding complexes of several secretory proteins in the ER. In our previous work, it was suggested that ERp29 function is critical for the folding/secretion of thyroglobulin (Tg), a major secretory product of thyroid cells. Current work is an attempt to substantiate this assumption by answering the question whether the secretion of Tg can be regulated through the manipulation of ERp29 expression in the FRTL-5 rat thyroid cells. Indeed, transient overexpression of ERp29 resulted in twofold enhancement of the Tg secretion whereas the RNAi-mediated ERp29 silencing led to the attenuation of the Tg export. Mutational analysis has suggested two loci that might be involved in the ERp29-Tg interactions: the interdomain linker including Cys157, an amino acid, which is important for the structural integrity of the C-terminal domain and an uncharged surface on the N-terminal domain flanked by Tyr64 and Gln70.