Analysis of protein transport through the Golgi in a reconstituted cell-free system.
The processes which transport membrane proteins between compartments of the Golgi apparatus have been reconstituted in vitro using isolated Golgi fractions. This cell-free system allows a detailed analysis of protein transport not possible in intact cells. Transport of the membrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is measured from a "donor" to an "acceptor" Golgi fraction. The donor Golgi fraction is prepared from VSV-infected Chinese hamster ovary (CHO) mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase I. "Acceptor" is prepared from uninfected wild-type CHO cells. Transport is measured by the addition of N-acetylglucosamine to G protein, which can occur only upon movement of G protein from donor to acceptor. Transport requires physiological pH and osmolarity, is dependent on nucleotide triphosphates, and is mediated by proteins both from cytosol and on the Golgi membranes. Protein movement is inhibited by the non-hydrolyzable GTP analogue, GTP gamma S. The process of transport proceeds through the budding, pinching off, targeting, and fusion of transport vesicles. In this system these vesicles are initially coated with a non-clathrin coat and are targeted with this coat intact. Several of the proteins which mediate transport have been characterized, and isolated to homogeneity. The successful development of this assay has led to the formulation of cell free assays for protein transport between other compartments. Comparison of these systems indicates that some common mechanisms of vesicular movement are used in transport between a variety of membrane compartments.
Wattenberg
B W
BW
Upjohn Company, Kalamazoo, Michigan 49001.
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation; D002474:Cell-Free System / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008561:Membrane Fusion / Q000502:physiology; D008562:Membrane Glycoproteins; D011506:Proteins / Q000378:metabolism*; D014759:Viral Envelope Proteins / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011506:Proteins; D014759:Viral Envelope Proteins; D019204:GTP-Binding Proteins