Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: role of the P58 containing compartment.
The glycoside digitonin was used to selectively permeabilize the plasma membrane exposing functionally and morphologically intact ER and Golgi compartments. Permeabilized cells efficiently transported vesicular stomatitis virus glycoprotein (VSV-G) through sealed, membrane-bound compartments in an ATP and cytosol dependent fashion. Transport was vectorial. VSV-G protein was first transported to punctate structures which colocalized with p58 (a putative marker for peripheral punctate pre-Golgi intermediates and the cis-Golgi network) before delivery to the medial Golgi compartments containing alpha-1,2-mannosidase II and processing of VSV-G to endoglycosidase H resistant forms. Exit from the ER was inhibited by an antibody recognizing the carboxyl-terminus of VSV-G. In contrast, VSV-G protein colocalized with p58 in the absence of Ca2+ or the presence of an antibody which inhibits the transport component NSF (SEC18). These studies demonstrate that digitonin permeabilized cells can be used to efficiently reconstitute the early secretory pathway in vitro, allowing a direct comparison of the morphological and biochemical events involved in vesicular tafficking, and identifying a key role for the p58 containing compartment in ER to Golgi transport.
Plutner
H
H
Department of Cell and Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Davidson
H W
HW
Saraste
J
J
Balch
W E
WE
D000251:Adenosine Triphosphatases; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification; D002451:Cell Compartmentation; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured / Q000166:cytology; D004072:Digitonin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000276:immunology / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism*; D008361:Mannosidases / Q000302:isolation & purification; D008561:Membrane Fusion / Q000187:drug effects / Q000276:immunology; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins / Q000302:isolation & purification / Q000378:metabolism*; D043323:alpha-Mannosidase
D002352:Carrier Proteins; D005656:Fungal Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D014759:Viral Envelope Proteins; D004533:Egtazic Acid; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D008361:Mannosidases; D043323:alpha-Mannosidase; D000251:Adenosine Triphosphatases; C064404:SEC18 protein, S cerevisiae; D004072:Digitonin