RESUMO
Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/análise , Isomerases , Diester Fosfórico Hidrolases/análise , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Linhagem Celular , DNA Complementar/metabolismo , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Cinética , Metionina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosfatidilinositol Diacilglicerol-Liase , Fosfoinositídeo Fosfolipase C , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Plasmocitoma , Isomerases de Dissulfetos de Proteínas , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
Protein disulfide isomerase (PDI), a luminal enzyme of the endoplasmic reticulum (ER), is thought to be involved in the process that assures that the correct disulfide bonds form as a newly synthesized protein folds into its appropriate three-dimensional structure (Freeman, 1984). In recent years, the ER has been shown to have at least two additional, distinct PDI-related luminal proteins (Bennett et al., 1988; Mazzarella et al., 1990). As a potential first step toward an investigation of the structure and function of PDI and of the PDI-related proteins as well, we have developed a bacterial expression system in Escherichia coli capable of synthesizing significant levels of enzymatically active PDI under the control of the inducible tac promoter. We have observed that the use of this bacterial expression system is complicated by the fact that there is a significant amount of internal initiation of protein synthesis within the PDI coding sequence and the fact that all of the PDI-related expression products are found equally distributed between the cytoplasmic and periplasmic fractions due to a single peptide-independent mechanism. Our studies with this system have demonstrated that at least some truncated PDI molecules containing the carboxy-terminal most active site have significant PDI activity.
Assuntos
Isomerases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Escherichia coli/genética , Expressão Gênica , Isomerases/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Isomerases de Dissulfetos de Proteínas , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Protein disulfide isomerase (PDI, ERp59), ERp72, and ERp61 are luminal proteins of the endoplasmic reticulum (ER) that are characterized by the presence of sequences corresponding to the active site regions of PDI. Each one of these proteins possesses a different COOH-terminal tetrapeptide ER retention signal. In order to investigate what other tetrapeptide sequences could serve as retention signals and to determine to what extent the function of the retention signal is modulated by the protein carrying the signal, we have constructed a set of mutants of two of these resident ER proteins, PDI and ERp72. In each of these proteins, the wild type tetrapeptide sequences were replaced by each member of the set of the 12 possible combinations consisting of (K,R,Q)-(D,E)-(D,E)-L. Analysis of the efficiency of retention of the variant proteins when each was transiently expressed in COS cells showed that the retention efficiencies vary with both the COOH-terminal sequence and with the protein that carries this sequence.
Assuntos
Retículo Endoplasmático/metabolismo , Isomerases/genética , Glicoproteínas de Membrana/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese , Isomerases de Dissulfetos de ProteínasRESUMO
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.