No abstract

The Golgi complex consists of a series of stacked cisternae in most eukaryotes. Morphological studies indicate the existence of intercisternal cross-bridge structures that may mediate stacking, but their identity is unknown. We have identified a 400-kDa protein, giantin, that is localized to the Golgi complex because its staining in double immunofluorescence experiments was coincident with that of galactosyltransferase, both in untreated cells and in cells treated with agents that disrupt Golgi structure. A monoclonal antibody against giantin yielded Golgi staining in one avian and all mammalian cell types tested, indicating that giantin is a conserved protein. Giantin exhibited reduced mobility on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was recovered in membrane fractions after differential centrifugation or sucrose flotation, and was not released from membranes by carbonate extraction. Thus, giantin appears to be an integral component of the Golgi membrane with a disulfide-linked lumenal domain. Strikingly, the majority of the polypeptide chain is cytoplasmically disposed, because large (up to 350 kDa) proteolytic fragments of giantin could be released from intact Golgi vesicles. This feature, a large contiguous cytoplasmic domain, is present in the calcium-release channel of muscle that cross-bridges the sarcoplasmic reticulum and transverse tubule membranes. Therefore, giantin's localization, conservation, and physical properties suggest that it may participate in forming the intercisternal cross-bridges of the Golgi complex.

Combined deficiency of factors V and VIII is an autosomal recessive bleeding disorder resulting from alterations in an unknown gene on chromosome 18q, distinct from the factor V and factor VIII genes. ERGIC-53, a component of the ER-Golgi intermediate compartment, was mapped to a YAC and BAC contig containing the critical region for the combined factors V and VIII deficiency gene. DNA sequence analysis identified two different mutations, accounting for all affected individuals in nine families studied. Immunofluorescence and Western analysis of immortalized lymphocytes from patients homozygous for either of the two mutations demonstrate complete lack of expression of the mutated gene in these cells. These findings suggest that ERGIC-53 may function as a molecular chaperone for the transport from ER to Golgi of a specific subset of secreted proteins, including coagulation factors V and VIII.

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIa (PI4K-IIIa) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIa, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain–specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed α-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into α-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.

No abstract

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145–3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40–70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical–basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37°C or in n-octyl-β-d-glycoside at 4°C (representative of GPIanchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+– K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.