Structure and assembly of the endoplasmic reticulum: biosynthesis and intracellular sorting of ERp61, ERp59, and ERp49, three protein components of murine endoplasmic reticulum.
Rabbit antibodies have been prepared against ERp61, ERp59, and ERp49, three protein components of rough endoplasmic reticulum (RER) purified from mineral oil-induced plasmacytoma 315 (MOPC-315) tissue. Analysis of subcellular fractions of MOPC-315 tissue by an immunoprecipitation procedure demonstrated that all three endoplasmic reticulum proteins (ERps) were most enriched in the RER. Immunologically cross-reacting proteins of similar molecular weight have been detected in other eucaryotic cell lines. We have used these antibodies to study the post-translational processing and biosynthetic sorting of the three ERps in pulse-labeled MOPC-315 cells. No larger precursor forms of the ERps were detected and none of the ERps were found to possess asparagine-linked oligosaccharide moieties. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 cells to study the biosynthetic sorting of ERp61, ERp59 and ERp49 and have found no evidence to suggest that these proteins ever leave the endoplasmic reticulum. In addition, all three ERps appeared to have luminally exposed domains. ERp61 and ERp59 were entirely protected by the ER membrane in the absence of detergent, while ERp49 was a transmembrane protein that also possesses a cytoplasmically exposed domain. We have used the anti-ERp antibodies to quantitate the synthesis and accumulation of the three ERps during lipopolysaccharide (LPS)-induced lymphocyte differentiation. After 48 h of culture in the presence of LPS, the synthesis of ERp49 increased sixfold relative to that in control cells. The synthesis and membrane accumulation of ERp61 and ERp59 were less affected by the LPS treatment. Thus, membranes isolated from LPS-treated cells were enriched in ERp49 relative to those isolated from control cells.
D000818:Animals; D000918:Antibody Specificity; D002460:Cell Line; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007120:Immunochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D010954:Plasmacytoma; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
D008565:Membrane Proteins