The soluble, calcium-binding protein calreticulin shares high sequence homology with calnexin, a transmembrane chaperone of glycoprotein folding. Our experiments demonstrated that calreticulin, like calnexin, associated transiently with numerous newly synthesized proteins in the endoplasmic reticulum. The population of proteins that bound to calreticulin was partially overlapping with those that bound to calnexin. Hemagglutinin (HA) of influenza virus was shown to associate with both calreticulin and calnexin. Using HA as a model substrate, it was found that both calreticulin- and calnexin-bound HA corresponded primarily to incompletely disulfide-bonded folding intermediates and conformationally trapped forms. Binding of all substrates was oligosaccharide-dependent and required the trimming of glucose residues from asparagine-linked core glycans by glucosidases I and II. In vitro, alpha-mannosidase digestion of calreticulin-bound HA indicated that calreticulin was specific for monoglucosylated glycans. Thus, calreticulin appeared to be a lectin with similar oligosaccharide specificity as its membrane-bound homologue, calnexin. Both are therefore likely to play an important role in glycoprotein maturation and quality control in the endoplasmic reticulum.

We report on the isolation, sequence and a putative role of a human endoplasmic-reticulum-lumenal protein, ERp28. The protein has the C-terminal retention signal KEEL and localizes to the endoplasmic reticulum (ER) as seen by subcellular fractionation and immunofluorescence studies. The protein has significant sequence similarity to members of the protein disulfide isomerase (PDI) family, although it lacks the thioredoxin box (CGHC) motif. We propose, on the basis of sequence analysis, a model of the domain structure of PDI, representing a significant extension of previously proposed models. Our results are in partial agreement with recently published NMR data [Kemmink, J., Darby, J., Dijkstra, K., Nilges, M. & Creighton, T. E. (1997) Curr. Biol. 7, 239Ϫ245] and indicate that PDI contains, in addition to the two thioredoxin folds described in previous models, two thioredoxin folds within the domains previously defined as b and b′. The thioredoxin domain of ERp28 shares a higher degree of similarity with the corresponding active and inactive domains of PDI than with other members of the PDI family, indicating that ERp28 developed from an ancient form of PDI or a PDI precursor. In contrast to Ig-heavy-chainbinding protein, human ERp28 is not induced by metabolic stress (tunicamycin). In in vitro experiments, ERp28 and calnexin precipitate with overexpressed, wild-type hepatitis B small surface antigen and with a mutated ER-retained form. This indicates that ERp28, as calnexin, may be involved in the processing of secretory proteins within the ER.Keywords : ERp28; ERp29; protein disulfide isomerase ; thioredoxin; endoplasmic reticulum.The endoplasmic reticulum (ER) is a specialized compartment within eucaryotic cells, in which many posttranslational modifications of proteins destined for secretion or targeting to other organelles are initiated or completed. Among these modifications are protein folding and oligomerization, catalyzed in part by the enzymes prolyl-peptidyl-cis-trans-isomerase [1], Igheavy-chain-binding protein (BiP, a eucaryotic heat-shock-protein-70-related protein) [2,3], and protein disulfide isomerase [4Ϫ6]. PDI is the most prominent member of a growing family of related proteins, the PDI-like family of protein oxidoreductases [7,8]. Characteristic of these proteins is the presence of a conserved stretch of amino acids with high sequence similarity to the cytoplasmic enzyme thioredoxin [9]. These similarities flank the tetrapeptide CXXC, a sequence that is known as the thioredoxin-box motif. The thioredoxin-box motif occurs twice in PDI, the closely related ER60 proteins [10] identity between these proteins is low. The roles of these proteins in the ER are poorly understood, though they may be involved in protein folding, functioning as oxidoreductases in the formation/isomerization of disulfide bonds and/or as molecular chaperones with peptide binding and folding activity independent of the reactive cysteines [16Ϫ19]. The group of Sitia [20] has shown that many of the ER-resident proteins may...

Rats treated with monocrotaline (MCT) develop pulmonary hypertension. Their right ventricles (RVs) exhibit severe pressure overload-induced hypertrophy, whereas the left ventricles (LVs) are normally loaded. In contrast, enhanced neuroendocrine stimulation during the transition to heart failure affects both ventricles. We assessed gene expression levels of Ca2+-regulating proteins in RVs and LVs of control and MCT rats in transition to heart failure to identify biomechanical load-regulated genes. In MCT RVs, both mRNA and protein levels of the Ca2+-ATPase of the sarcoplasmic/endoplasmic reticulum (SERCA2a) were reduced by 36% (P=0.001) and 17% (P=0.016), respectively, compared with control RVs. Phospholamban and ryanodine receptor mRNA levels likewise were reduced (by 27% [P=0.05] and 21% [P=0.011], respectively) in MCT RVs, whereas sarcolemmal Na+-Ca2+ exchanger expression was not altered. MCT LVs exhibited no significant expression changes compared with control LVs. Isometrically contracting MCT intact RV trabeculae showed enhanced baseline force development. Although control RV preparations exhibited a positive force-frequency relationship, MCT RVs showed a negative force-frequency relationship and blunted postrest potentiation. Contractile function of MCT LV trabeculae was normal. Maximum Ca2+-activated tension was enhanced by 64% in permeabilized RV MCT preparations (P=0.013). beta-Myosin heavy chain protein was upregulated in MCT RVs (P<0.001) but unaltered in MCT LVs. Degradation of troponin T was prominent in MCT RVs, a phenomenon not observed in the LV. Enhanced biomechanical load is necessary to induce the gene expression changes associated with the hypertrophic phenotype of the pressure-overloaded RV. Neuroendocrine factors, which equally affect both chambers, are not sufficient to alter the expression of Ca2+-cycling proteins.

Protein II isolated from porcine intestinal epithelium is a Ca2+-modulated lipid-binding protein. The amino acid sequence of porcine protein II reported here sheds new light on the properties of a multigene protein family which includes the tyrosine kinase substrates of the sarc gene (p36) and of the EGF-receptor (p35). The sequence consolidates the structural principle in which an amino-terminal tailpiece of variable length is followed by a core built from four internally homologous segments for those proteins in the 35-40 kd range. Sequence data also show that the core can now be described as two domains each containing one low and one high homology segment. This view accounts for two Ca2+ sites, lipid aggregation and F-actin bundlingwhen present and suggests that properties of the cores in which protein II differs from p36 and p35 arise primarily from segments 1 and 2. The protease-sensitive tailpiece of protein II is very short and lacks the phosphorylatable tyrosine present in the larger tail domains of p36 and p35. It harbors, however, like the p36 domain, the major site for in vitro phosphorylation by the Ca2+and lipid-activated protein kinase C. In protein H this site is most likely threonine 6. The sequence alignment also explains why protein II does not interact with a unique pll, a property probably specific for p36. Our results further suggest that liver endonexin may reflect two protein species both closely related to protein II.

P36 was originally defined as the major cytoplasmic target of retrovirally coded tyrosine‐kinases. While recently much has been learned about its biochemistry, the functional importance of its tyrosine and serine phosphorylation has not been approached. As p36 is now understood as a multi‐ligand protein its in vitro phosphorylation by three different serine/threonine kinases was followed. Monomeric p36 is a much better substrate than the complex containing two copies each of p36 and p11 (protein I). All p36 phosphorylation sites occur within the amino‐terminal 29 residues specifically released by mild proteolysis. As this region harbors an important interaction site for p11 the reduced phosphorylation of p36 in the protein I complex results most likely from a lowered accessibility. Phosphorylation of p36 is serine specific. Reconstitution experiments define at least two functionally distinct sites. One product of protein kinase C reconstitutes with p11 to protein I, while this complex formation normal for p36 is observed neither for the second phosphorylation product nor for the derivatives resulting from phosphorylation by calmodulin or cAMP dependent kinases. The results lend direct support to the hypothesis that phosphorylation of p36 can modulate one of its molecular functions. Obvious implications for other Ca2+‐dependent lipid binding proteins are discussed.