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 Man(8-9) to Man(5) processing

We report the reconstitution of the transfer of a membrane glycoprotein (ve sicular stomatitis virus glycoprotein, VSV-G protein) from endoplasmic reti culum to Golgi apparatus and its subsequent Man(8-9)GlcNAc(2) to Man(5)GlcN Ac(2) processing in a completely cell-free system. The acceptor was Golgi a pparatus from rat liver immobilized on nitrocellulose, The endoplasmic reti culum donor was from homogenates of VSV-G-infected BHK cells. Nucleoside tr iphosphate plus cytosol-dependent transfer and processing of radiolabeled V SV-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 trans ferred was processed to the Man(5)GlcNAc(2) form. Processing was specific f or cis Gels apparatus fractions purified by preparative free-flow electroph oresis, Fractions derived from the trans Golgi apparatus mere inactive in p rocessing, With the ts045 temperature-sensitive mutant, transfer and proces sing were much reduced even in the complete system when microsomes were fro m cells infected with mutant virus and incubated at the restrictive tempera ture of 39.5 degrees C but were able to proceed at the permissive temperatu re of 34 degrees C. Thus, Man(8-9)GlcNAc(2) to Man(5)GlcNAc(2) processing o f VSV-G protein occurs following transfer in a completely cell-free system using immobilized intact Golgi apparatus or cis Golgi apparatus cisternae a s the acceptor and shows temperature sensitivity, donor specificity, requir ement for ATP, and response to inhibitors similar to those exhibited by tra nsfer and processing of VSV-C; protein in vivo. (C) 1999 Academic Press.