Chlamydiae are obligate intracellular pathogens that spend their entire growth phase sequestered in a membrane-bound vacuole called an inclusion. A set of chlamydial proteins, labelled Inc proteins, has been identified in the inclusion membrane (IM). The predicted IncA, IncB and IncC amino acid sequences share very limited similarity, but a common hydrophobicity motif is present within each Inc protein. In an effort to identify a relatively complete catalogue of Chlamydia trachomatis proteins present in the IM of infected cells, we have screened the genome for open reading frames encoding this structural motif. Hydropathy plot analysis was used to screen each translated open reading frame in the C. trachomatis genome database. Forty-six candidate IM proteins (C-lncs) that satisfied the criteria of containing a bilobed hydrophobic domain of at least 50 amino acids were identified. The genome of Chlamydia pneumoniae encodes a larger collection of C-lnc proteins, and only approximately half of the C-lncs are encoded within both genomes. In order to confirm the hydropathy plot screening method as a valid predictor of C-lncs, antisera and/or monoclonal antibodies were prepared against six of the C. trachomatis C-lncs. Immunofluorescence microscopy of C. trachomatis-infected cells probed with these antibodies showed that five out of six C-lncs are present in the chlamydial IM. Antisera were also produced against C. pneumoniae p186, a protein sharing identity with Chlamydia psittaci lncA and carrying a similar bilobed hydrophobic domain. These antisera labelled the inclusion membrane in C. pneumoniae infected cells, confirming that proteins sharing the unique secondary structural characteristic also localize to the inclusion membrane of C. pneumoniae. Sera from patients with high-titre antibodies to C. trachomatis were examined for reactivity with each tested C-lnc protein. Three out of six tested C-lncs were recognized by a majority of these patient sera. Collectively, these studies identify and characterize novel proteins localized to the chlamydial IM and demonstrate the existence of a potential secondary structural targeting motif for localization of chlamydial proteins to this unique intracellular environment.
We have characterized the biosynthesis and intracellular transport of a membrane glycoprotein, designated plgp57, which is found predominantly in the prelysosome compartment (PLC) of Madin-Darby bovine kidney cells. In pulse-chase experiments, plgp57 was found to be initially synthesized as a 35 kDa precursor which was modified to yield a diffuse approximately 57 kDa mature form. Digestion with endoglycosidase H (endo H) demonstrated that the 35 kDa precursor contained three endo H-sensitive, high mannose-type oligosaccharides which became modified to endo H-resistant, complex-type sugars on the approximately 57 kDa mature form. Labelling cells in the presence of tunicamycin and treatment of the 35 kDa precursor with endo H revealed that plgp57 has a core protein of 24 kDa to which three N-asparagine-liked oligosaccharides are attached. Other experiments indicated that plgp57 could be differentially glycosylated on a common 24 kDa core protein in different cell types. The half-lives of the glycosylated and non-glycosylated forms of plgp57 were approximately 18 and approximately 13 h, respectively. Glycosylated and non-glycosylated plgp57 exhibited similar steady-state intracellular distributions, indicating that targeting of plgp57 to the PLC does not require carbohydrate address markers. Pulse-labeling of cells followed by organelle fractionation at various chase times revealed a t1/2 = approximately 1 h for the transit of newly synthesized plgp57 to the PLC. Finally, amino terminal sequencing of plgp57 revealed the similarity of this protein to the CD63/ME491 family of membrane glycoproteins.
The compositional relationship between the cell surface of rabbit polymorphonuclear leukocytes (PMNs) and the membranes of PMN cytoplasmic granules has been investigated. Heterophilic PMNs obtained from peritoneal exudates contained 13 cell surface polypeptides ranging in molecular weight from 220,000 to 12,000 daltons as determined by lactoperoxidase-catalyzed protein iodination and gel electrophoresis. Of these, four polypeptides co-migrated with proteins identified as the major constituents of specific (SpG) and azurophilic (AzG) granule membranes. The most notable of these were cell surface proteins of 145,000 and 96,000 daltons that co-migrated with SpG membrane proteins and a 48,000-dalton protein that was also a major component of AzG membranes. Also, four iodinated cell surface proteins co-migrated with proteins identified as granule content proteins released from PMNs during exocytosis. Extensive washing did not remove these proteins from the cell surface.Iodination of PMNs after the release of SpG and AzG contents by calcium ionophore-induced exocytosis revealed that there was not a dramatic qualitative change in the proteins on the cell surface. Instead, there were large, quantitative increases in the relative amounts of 1251 that were incorporated into several pre-existing cell surface proteins; all of these cell surface proteins co-migrated as a set with those polypeptides identified as either granule membrane or content proteins. Although nearly all of the major polypeptides of SpG and AzG had counterparts on the cell surface of freshly isolated peritoneal exudate PMNs, there were several polypeptides that were unique to the cell surface. Thus, the PMN has at least three membrane compartments with strikingly different protein compositions.Numerous morphological studies have demonstrated that in secretory cells membrane-bounded granules fuse with the plasma membrane during secretion (exocytosis), resulting in the mixing of these membranous compartments (t). In addition, recent evidence suggests that components of secretory cell plasma membranes may recycle back to the Golgi complex to be reutilized for secretory granule formation (2, 3). Moreover, there are indications that in some cells newly added secretory granule membranes may recycle, intact, from the plasma membrane back to the cytoplasm (4). Even with this information, the compositional relationship between the plasmalemma and secretory granule membranes is not well documented, owing to the difficulty in obtaining plasma and granule membranes from the same cell type. Rabbit polymorphonuclear heterophils (PMNs), since they exist as single cells in vivo, possess a readily accessible plasma membrane, two well-defined populations of cytoplasmic granules, and a capacity for secreting the contents of these granules into the extraceUular space. Thus, these cells provide an excellent model for studying the relationship between the plasma membrane and secretory granule membranes.In the preceding paper (5) we reported on the separation of rabb...
It has been proposed that polymeric IgA is translocated from plasma to bile across hepatocytes of the rat liver by a secretory component-mediated, vesicular transport. To define the ultrastructural details of the proposed transport mechanism, we employed peroxidase-labeled antibody immunocytochemistry to localize secretory component in the rat liver and monitor the hepatic translocation of homologous myeloma polymeric IgA infused i.v. Secretory component was found associated with the endoplasmic reticulum, Golgi complexes, cytoplasmic vesicles, and plasma membranes of the sinusoidal and canalicular surfaces of hepatocytes; secretory component at the sinusoidal surface was most prominent in micropinocytic invaginations or pits. Livers were examined for the sites of polymeric IgA 5, 15, and 30 min after infusion. Evidence was obtained that polymeric IgA is translocated across hepatocytes by a series of events: 1) polymeric IgA binds selectively to secretory component on the external surface of the sinusoidal plasma membrane; 2) secretory component-IgA complexes are internalized in endocytic vesicles; 3) the vesicles migrate through the cytoplasm without association with lysosomes or Golgi complexes; 4) the vesicles fuse with the cytoplasmic surface of the bile canalicular membrane, where secretory component-IgA complexes are released into bile by exocytosis.
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