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PMID: 2125301 Published · ppublish English Journal Article

Transport of influenza HA from the trans-Golgi network to the apical surface of MDCK cells permeabilized in their basolateral plasma membranes: energy dependence and involvement of GTP-binding proteins.

The Journal of cell biology ·Vol. 111 ·No. 6 Pt 2 ·1990-12-00 ·Pages 2893-908

Gravotta D, Adesnik M, Sabatini DD

Abstract

A procedure employing streptolysin O to effect the selective permeabilization of either the apical or basolateral plasma membrane domains of MDCK cell monolayers grown on a filter support was developed which permeabilizes the entire monolayer, leaves the opposite cell surface domain intact, and does not abolish the integrity of the tight junctions. This procedure renders the cell interior accessible to exogenous macromolecules and impermeant reagents, permitting the examination of their effects on membrane protein transport to the intact surface. The last stages of the transport of the influenza virus hemagglutinin (HA) to the apical surface were studied in pulse-labeled, virus-infected MDCK cells that were incubated at 19.5 degrees C for 90 min to accumulate newly synthesized HA in the trans-Golgi network (TGN), before raising the temperature to 35 degrees C to allow synchronized transport to the plasma membrane. In cells permeabilized immediately after the cold block, 50% of the intracellular HA molecules were subsequently delivered to the apical surface. This transport was dependent on the presence of an exogenous ATP supply and was markedly inhibited by the addition of GTP-gamma-S at the time of permeabilization. On the other hand, the GTP analogue had no effect when it was added to cells that, after the cold block, were incubated for 15 min at 35 degrees C before permeabilization, even though at this time most HA molecules were still intracellular and their appearance at the cell surface was largely dependent on exogenous ATP. These findings indicate that GTP-binding proteins are involved in the constitutive process that effects vesicular transport from the TGN to the plasma membrane and that they are charged early in this process. Transport of HA to the cell surface could be made dependent on the addition of exogenous cytosol when, after permeabilization, cells were washed to remove endogenous cytosolic components. This opens the way towards the identification of cell components that mediate the sorting of apical and basolateral membrane components in the TGN and their polarized delivery to the cell surface.

MeSH Terms
Animals Bacterial Proteins Biological Transport, Active/drug effects,physiology Cell Membrane Permeability Cells, Cultured GTP-Binding Proteins/physiology Golgi Apparatus/metabolism Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology Guanosine Triphosphate/physiology Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral/biosynthesis,metabolism Membrane Proteins/metabolism Models, Biological Streptolysins Viral Envelope Proteins/metabolism
Chemicals
Bacterial Proteins Hemagglutinin Glycoproteins, Influenza Virus Hemagglutinins, Viral Membrane Proteins Streptolysins Viral Envelope Proteins streptolysin O Guanosine 5'-O-(3-Thiotriphosphate) Guanosine Triphosphate GTP-Binding Proteins
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Gravotta D
Department of Cell Biology, New York University Medical Center 10016.
Adesnik M
Sabatini D D
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Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.111.6.2893
Published
1990-12-00
Pages
2893-908
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2116360
Subset
IM
External Links
PubMed source DOI: 10.1083/jcb.111.6.2893 PMC Full Text View journal details
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