The role of vacuolar H+-ATPase in exocytic and endocytic membrane transport processes
|Organizations:||University of Oulu, Faculty of Medicine, Department of Anatomy and Cell Biology
University of Oulu, Biocenter Oulu
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|Persistent link:|| http://urn.fi/urn:isbn:9514252764
|Publish Date:|| 1999-06-01
|Thesis type:||Doctoral Dissertation
|Defence Note:||Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in the Auditorium of the Department of Anatomy, on June 18th, 1999, at 12 noon.
Professor Marja Makarow
Docent Antero Salminen
The role of vacuolar H+-ATPase (V-ATPase) in exocytic and endocytic membrane transport processes was studied by using its specific inhibitor, bafilomycin A1 (Baf A1), as a tool. On the exocytic pathway, both brefeldin A- and nocodazole-induced retrograde transport of Golgi proteins to the endoplasmic reticulum (ER) were inhibited by Baf A1. Furthermore, p58/ERGIC-53, which normally cycles between the ER, the intermediate compartment (IC), and cis-Golgi, was arrested in pre-Golgi tubules and vacuoles, and the number of p58-positive 80-nm Golgi (COPI) vesicles was reduced, suggesting that the drug inhibits the vesicle-mediated retrieval of the protein from post-ER compartments. The small GTPase rab1p was efficiently recruited to the tubules, accumulating in the presence of Baf A1. In contrast, these tubules showed no enrichment of anterogradely transported proteins, indicating that they participate in retrograde transport. Interestingly, acidic lumenal pH could only be detected in the more central pre-Golgi elements.
The forward (anterograde) transport of newly synthesized Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV) glycoproteins from the ER to the cis-Golgi was largely unaffected by Baf A1. However, maturation processes occurring in the trans-Golgi were inhibited, and the amounts of viral glycoproteins appearing at the cell surface were reduced. Newly synthesized VSV glycoprotein accumulated into rab1p-positive Golgi membranes in the presence of Baf A1, indicating that the transport from cis-Golgi was affected. Furthermore, O-glycosylation of the expressed CD8 chimeras and lectin cytochemistry experiments indicate that Baf A1 affects the transport from cis-Golgi. Instead, Baf A1 did not affect the transport of viral glycoproteins from the trans-Golgi network to the cell surface. We propose, that anterograde intra-Golgi traffic may be affected indirectly by Baf A1, as it inhibits retrograde vesicle-mediated transport and thus cisternal maturation.
Baf A1 inhibited the entry of SFV into BHK-21 cells. Thus, V-ATPase was responsible for the acidification of the endosomes needed for virus entry. In cells infected with VSV and subsequently treated with Baf A1, virus particles were found to be accumulated in tubular membrane structures, which also contained endocytosed BSA-gold. Neither VSV nor BSA-gold particles were detected in lysosomal glycoprotein (lgp) 120-positive lysosomes, however. Thus, secreted and further endocytosed virus particles accumulate into tubulated endocytic organelles, apparently early endosomes, in Baf A1-treated cells. We conclude that the transport from endosomes to lysosomes is inhibited by Baf A1.
The bulk of rab7 GTPase, which participates in vesicle fusion to late endosomes, was localized to the ruffled border (RB) membrane of bone-resorbing osteoclast. This indicates that the membrane has some characteristics of late endosomal membranes and that endocytic membrane transport is oriented towards the RB. Consistently, both endocytosed lumenal horseradish peroxidase and receptor-bound transferrin were delivered to the RB. The delivery of membrane-associated transferrin to the RB further indicates that the RB has some endosomal characteristics and suggests that the endocytic pathway contributes to the maintenance of functional RB. The endocytic pathway could act in balancing the membrane traffic associated with transcytosis from the RB to the basal plasma membrane. Endocytic processes in osteoclasts appeared to be very sensitive to Baf A1. Thus, blocking of the endocytic membrane traffic towards the RB could explain the inactivation of cells by low concentrations of the drug.
Acta Universitatis Ouluensis. D, Medica
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