Cell-free transfer of the vesicular stomatitis virus G protein from an endoplasmic reticulum compartment of baby hamster kidney cells to a rat liver Golgi apparatus compartment for Man8-9 to Man5 processing.
We report the reconstitution of the transfer of a membrane glycoprotein (vesicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reticulum to Golgi apparatus and its subsequent Man8-9GlcNAc2 to Man5GlcNAc2 processing in a completely cell-free system. The acceptor was Golgi apparatus from rat liver immobilized on nitrocellulose. The endoplasmic reticulum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside triphosphate plus cytosol-dependent transfer and processing of radiolabeled VSV-G protein was observed with donor from BHK cells infected at 37 degrees C with wild-type VSV or at the permissive temperature of 34 degrees C with the ts045 mutant. With Golgi apparatus as acceptor, specific transfer at 37 degrees C in the presence of nucleoside triphosphate was eightfold that at 4 degrees C or in the absence of ATP. About 40% of the VSV-G protein transferred was processed to the Man5GlcNAc2 form. Processing was specific for cis Golgi apparatus fractions purified by preparative free-flow electrophoresis. Fractions derived from the trans Golgi apparatus were inactive in processing. With the ts045 temperature-sensitive mutant, transfer and processing were much reduced even in the complete system when microsomes were from cells infected with mutant virus and incubated at the restrictive temperature of 39.5 degrees C but were able to proceed at the permissive temperature of 34 degrees C. Thus, Man8-9GlcNAc2 to Man5GlcNAc2 processing of VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae as the acceptor and shows temperature sensitivity, donor specificity, requirement for ATP, and response to inhibitors similar to those exhibited by transfer and processing of VSV-G protein in vivo.
Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana, 47907, USA.
M A
MA
Paulik
C C
CC
Widnell
P A
PA
Whitaker-Dowling
N
N
Minnifield
D M
DM
Morré
D J
DJ
Morré
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005260:Female; D006023:Glycoproteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006596:Hexosaminidases / Q000378:metabolism; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D013696:Temperature; D014759:Viral Envelope Proteins / Q000378:metabolism*
C028391:G protein, vesicular stomatitis virus; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D000255:Adenosine Triphosphate; D006596:Hexosaminidases