Abstract
We have cloned, sequenced, and expressed full length cDNA clones encoding two abundant, luminal endoplasmic reticulum proteins (ERp), ERp59/PDI and ERp72. ERp59/PDI has been identified as the microsomal enzyme protein disulfide isomerase (PDI). An analysis of the amino acid sequence of ERp72 showed that it shared sequence identity with ERp59/PDI at three discrete regions, having three copies of the sequences that are thought to be the CGHC-containing active sites of ERp59/PDI. Thus, ERp72 appears to be a newly described member of the family of CGHC-containing proteins. ERp59/PDI has the sequence KDEL at its COOH terminus while ERp72 has the related sequence KEEL. Removal of the KDEL of ERp59/PDI or the KEEL of ERp72 by in vitro mutagenesis techniques and subsequent analysis of the mutants in transient expression assays, showed that both sequences are endoplasmic reticulum retention signals for their respective proteins. The most dramatic difference in secretion between the wild type and the mutant forms of the protein was seen in the case of ERp72.
Highlights
We have cloned, sequenced, and expressed full length cDNA clones encoding two abundant, luminal endoplasmic reticulum proteins (ERp), ERp59/protein disulfide isomerase (PDI) and ERp72
Identification, Expression and Sequencing of Full Length cDNA Clones for ERp59/PDI and ERp72--In earlier experiments, ERp59/PDI and ERp72 cDNA clones were identified by using hybrid selection techniques to screen a cDNA library prepared in pBR322 from a 15-19 S fraction of murine plasmacytoma poly(A)+ mRNA
The rabbit anti-murine ERp59/PDI and antimurine ERp72 used in these experiments and in the subsequent experiments discussed in this report had been extensively characterized in previous work (Lewis et al, 1985a, 1985b, and 1986)
Summary
ERp59/PDI and murine ERp72 to identify the cell-free translation products. The anti-ERp antisera had been extensively characterized in previous work (Lewis et aZ., 1985a, 1985b, and 1986). Clones-The full length ERp59/PDI and ERp72 pcD clones as well as the COOH-terminal deletion retention signal mutants were screened for protein expression by transfection into COS cells as described by Mazzarella and Green (1987), except that in all experiments the labeling media contained dialyzed fetal calf serum at a final concentration of 5%. This was done to ensure optimal secretory activity. The immunoprecipitates were analyzed by SDS-PAGE, and the autoradiograms were quantitated by densitometry using an LKB laser densitometer Ultrascan (model Z-102) coupled to an automatic integrator (LKB model 220 Bromma)
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