A novel pancreas-specific serpin (ZG-46p) localizes to the soluble and membrane fraction of the Golgi complex and the zymogen granules of acinar cells.

By immunoscreening a cDNA expression library of rat pancreas using a polyspecific antiserum raised against purified pancreatic zymogen granule membranes, we have identified a cDNA clone coding for a novel protein, named ZG-46p. The cDNA contains an ORF of 1215 bp coding for a protein of 405 amino acids with a calculated molecular mass of 46 kDa. Sequence analysis revealed high homologies to known serine protease inhibitors (serpins), e.g. human anti-thrombin III (47.2%) or human and rat anti-trypsin (44%). The highest homology is present in the serpin signature, a consensus sequence common to all serpins, as well as its flanking hinge region. Northern blot analysis reveals the exclusive expression of the novel serpin mRNA in the pancreas, both during embryonic development and in the adult gland but not in the acinar carcinoma cell line AR4-2J. In vitro translation experiments demonstrate that the protein is N-glycosylated, but in vivo and in vitro phosphorylation was not found in spite of multiple phosphorylation sites. By immunofluorescence studies pancreatic serpin was localized predominantly to the Golgi complex in a similar distribution as the marker protein TGN38. Western blot analysis of various subcellular fractions showed ZG-46p mainly as soluble protein in the Golgi but also in zymogen granule content. A minor but significant portion of the protein was firmly attached to both the zymogen granule and Golgi membranes as Triton X-114 extraction indicates. The cellular localization, the distribution in the soluble and membrane fraction of Golgi complex and zymogen granules, and the finding that pancreatic serpin is associated with aggregated secretory proteins suggest a role in the sorting of pancreatic enzymes during granule formation.

cDNA cloning and characterization of a novel 16 kDa protein located in zymogen granules of rat pancreas and goblet cells of the gut.

By immunoscreening of a cDNA expression library of rat pancreas with a polyspecific antibody to purified rat zymogen granule membranes, we have cloned a cDNA coding for a novel protein of about 16 kDa (ZG-16p). By both immunocytochemistry and Western blot analysis of different fractions of rat pancreas with anti-ZG-16 antibodies, the protein could be localized in the content fraction of zymogen granules and also, to a lesser extent, bound to the granule membranes. Computer-based sequence analysis revealed no significant homologies to any of the known proteins of zymogen granules. A N-terminal portion of about 20 amino acids was predicted as a potential secretory signal sequence and may reflect the intracellular localization of the protein. As revealed by Northern blot analysis of total RNA from various organs of the rat, expression of the corresponding gene is restricted by only pancreas, colon, duodenum, and, to a much lesser extent, stomach. No traces of ZG-16 RNA were detectable in any of the other tissues tested so far. Expression of the ZG-16-gene in pancreatic cells is slightly stimulated by treatment of rats with cerulein, a decapeptide analogue of cholecystokinin, which is known to stimulate secretion in acinar cells. In contrast, treatment of the rat pancreatic tumor cell line, AR4-2J, with 10 nM dexamethasone, which has been shown to increase the synthesis and secretion of all secretory enzymes of rat pancreas accompanied with an increase in the secretory machinery, leads to a remarkable increase in the expression of ZG-16. Thus the expression pattern of the ZG-16 gene in response to hormonal stimulation of rat pancreatic acinar cells resembles those found for most of the secretory enzymes. The localization of ZG-16p and the regulation of ZG-16 gene expression in response to hormonal stimulation of pancreatic acinar cells leads us to presume that this novel protein has a functional role in the complex and ill-understood processes involved in the regulated secretory pathway of these cells.

Distribution of the early light-inducible protein in the thylakoids of developing pea chloroplasts.

The distribution of the early light-inducible protein (ELIP) of pea (Pisum sativum) between grana and stroma thylakoids was studied. An antibody raised against a bacterial-expressed fusion protein containing ELIP sequences was used. Illumination of dark-grown pea seedlings causes an accumulation of the ELIP in the thylakoid membranes with a maximum level at 16 h. During continuous illumination exceeding 16 h the level decreases again. The fractionation of thylakoid membranes of 48-h-illuminated pea seedlings in grana and stroma thylakoids reveals that there is no uniform distribution of ELIP in the thylakoids. Rather 60-70% of ELIP was found in the stroma thylakoids and 30-40% in the grana thylakoids. This distribution is in accordance with that of photosystem I but not with that of photosystem II. After Triton-X-100 solubilization almost all ELIP is found in the photosystem-I-containing fraction. This also supports an association of ELIP with photosystem I.

Snurportin1, an m3G-cap-specific nuclear import receptor with a novel domain structure.

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-cap but not m7G-cap structures. Snurportin1 enhances the m3G-capdependent nuclear import of U snRNPs in both Xenopus laevis oocytes and digitonin-permeabilized HeLa cells, demonstrating that it functions as an snRNP-specific nuclear import receptor. Interestingly, solely the m3G-cap and not the Sm core NLS appears to be recognized by snurportin1, indicating that at least two distinct import receptors interact with the complex snRNP NLS. Snurportin1 represents a novel nuclear import receptor which contains an N-terminal importin beta binding (IBB) domain, essential for function, and a C-terminal m3G-cap-binding region with no structural similarity to the arm repeat domain of importin alpha.

The 20kD protein of human [U4/U6.U5] tri-snRNPs is a novel cyclophilin that forms a complex with the U4/U6-specific 60kD and 90kD proteins.

Cyclophilins (Cyps) catalyze the cis/trans isomerization of peptidyl-prolyl bonds, a rate-limiting step in protein folding. In some cases, cyclophilins have also been shown to form stable complexes with specific proteins in vivo and may thus also act as chaperone-like molecules. We have characterized the 20kD protein of the spliceosomal 25S [U4/U6.U5] tri-snRNP complex from HeLa cells and show that it is a novel human cyclophilin (denoted SnuCyp-20). Purified [U4/U6.U5] tri-snRNPs, but not U1, U2, or U5 snRNPs, exhibit peptidyl-prolyl cis/trans isomerase activity in vitro, which is cyclosporin A-sensitive, suggesting that SnuCyp-20 is an active isomerase. Consistent with its specific association with tri-snRNPs in vitro, immunofluorescence microscopy studies showed that SnuCyp-20 is predominantly located in the nucleus, where it colocalizes in situ with typical snRNP-containing structures referred to as nuclear speckles. As a first step toward the identification of possible targets of SnuCyp-20, we have investigated the interaction of SnuCyp-20 with other proteins of the tri-snRNP. Fractionation of RNA-free protein complexes dissociated from isolated tri-snRNPs by treatment with high salt revealed that SnuCyp-20 is part of a biochemically stable heteromer containing additionally the U4/U6-specific 60kD and 90kD proteins. By coimmunoprecipitation experiments performed with in vitro-translated proteins, we could further demonstrate a direct interaction between SnuCyp-20 and the 60kD protein, but failed to detect a protein complex containing the 90kD protein. The formation of a stable SnuCyp-20/60kD/90kD heteromer may thus require additional factors not present in our in vitro reconstitution system. We discuss possible roles of SnuCyp-20 in the assembly of [U4/U6.U5] tri-snRNPs and/or in conformational changes occurring during the splicing process.