Abstract. a-mannosidases I and II (Man I and 11) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis-and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man 1I was most commonly found in medial-and/or trans-cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO ceils), both medial and trans (pancreatic acinar cells, enterocytes), or trans-(goblet ceils) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medialand trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.
IntroductionGeneralized atrophic benign epidermolysis bullosa (GA-BEB) is a form of nonlethal junctional epidermolysis bullosa characterized by universal alopecia and atrophy of the skin. We report a deficiency of the 180-kD bullous pemphigoid antigen in three patients with GABEB from unrelated families. We screened specimens of clinically normal skin from nine junctional epidermolysis bullosa patients (3 GABEB, 4 lethal, 1 cicatricial, 1 pretibial) by immunofluorescence using monoclonal antibodies to the 180-kD and 230-kD bullous pemphigoid antigens (BP180 and BP230). In the skin of the three GABEB patients there was no reactivity with antibodies to BP180, whereas staining for BP230 was normal. In the skin of the other six, non-GABEB patients, included in this study the expression of BP180 and BP230 was normal. Immunoblot analysis of cultured keratinocytes from one of the GABEB patients also failed to detect BP180 antigen, whereas BP230 was present in normal amounts. In previous studies on the expression of the bullous pemphigoid antigen in JEB, sera from bullous pemphigoid (BP) patients were used, the specificity of which had not been analyzed by immunoblotting (6-9). The expression was found to be normal or variable. Later it became apparent that BP-serum is a mixture of polyclonal antibodies, which are very difficult to characterize, even after affinity-purification (10). Fortunately, reliable monospecific antibodies for each of the two major bullous pemphigoid antigens have now become available (1 1, 12).In this study we used monoclonal antibodies specific for BPI 80 and BP230 (11,12). In addition we studied the expression of the newly described 500-kD hemidesmosomal plaque protein HD1 (13)
The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.
The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.
We have examined the role played by protein kinase A (PKA) in vesicle-mediated protein transport from the trans-Golgi network (TGN) to the cell surface. In vivo this transport step was inhibited by inhibitors of PKA catalytic subunits (C-PKA) such as the compound known as H89 and a myristoylated form of the inhibitory peptide sequence contained in the thermostable PKA inhibitor. Inhibition by H89 occurred at an early stage during the transfer of vesicular stomatitis virus G glycoprotein from the TGN to the cell surface. Reversal from this inhibition correlated with a transient increase in the number of free coated vesicles in the Golgi area. Vesicle budding from the TGN was studied in vitro using vesicular stomatitis virusinfected, permeabilized cells. Addition to this assay of C-PKA stimulated vesicle release while it was suppressed by PKA inhibitory peptide, H89, and antibody against C-PKA. Furthermore, vesicle release was decreased when PKA-depleted cytosol was used and restored by addition of C-PKA. These results indicate a regulatory role for PKA activity in the production of constitutive transport vesicles from the TGN.Transport of newly synthesized proteins along the exocytic pathway is mediated by small vesicular carriers (1, 2). A set of proteins collectively known as the transport machinery is responsible for the formation and consumption of vesicles. These include receptor proteins that sort and concentrate cargo molecules at the budding site, coat proteins that catalyze budding from the donor compartment, and proteins responsible for targeting and fusion with the acceptor membrane (1). Recent evidences indicate that the transport machinery is regulated by specific phosphorylating activities that in this way modulate intracellular traffic. Thus, phosphatidylinositol-3 kinase has been shown to be required for transfer of hydrolases from the Golgi to either the yeast vacuole (3) or the mammalian lysosomes (4, 5). Release of secretory vesicles from the trans-Golgi network (TGN) of endocrine cells has also been shown to be regulated by tyrosine phosphorylation (6). Both protein kinase A (PKA) and protein kinase C (PKC) activities have been reported to control particular vesiclemediated transport steps such as constitutive (7-11) and regulated exocytosis (12-15) and transcytosis (16,17). Whereas the effects derived from the activation͞inhibition of these different kinases on transport are well documented little information is available about their mechanism of action. PKC activity has been described to stimulate association of ADP ribosylation factor and coatomer proteins to Golgi membranes (18) but recent data indicate that its phosphorylating activity is not required for vesicle budding from the TGN (19). Therefore, it is uncertain at present in which way protein kinases control transport activities. We have recently described that PKA differentially regulates constitutive protein transport along the exocytic pathway with transport from the TGN to the cell surface being strictly dependent on PKA ...
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