Abstract
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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-5276-4 |
| Date | 01 June 1999 |
| Creators | Palokangas, H. (Harri) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 1999 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3221, info:eu-repo/semantics/altIdentifier/eissn/1796-2234 |
Page generated in 0.0026 seconds