Reconstitution of Transport of Vesicular Stomatitis Virus G Protein from the Endoplasmic Reticulum to the Golgi Complex Using a Cell-free System

Transport of the vesicular stomatitis virusencoded glycoprotein (G protein) between the endoplasmic reticulum (ER) and the cis Golgi compartment has been reconstituted in a cell-free system. Transfer is measured by the processing of the high mannose (mans_gGlcNAc2) ER form of G protein to the mansGlcNAc2 form by the cis Golgi enzyme a-mannosidase I. G protein is rapidly and efficiently transported to the Golgi complex by a process resembling that observed in vivo. G protein is trimmed from the high mannose form to the mansGlcNAc2 form without the appearance of the intermediate man6_TGlcNAcz oligosaccharide species, as is observed in vivo. G protein is found in a sealed membrane-bound compartment before and after incubation. Processing in vitro is sensitive to detergent, and the Golgi ~t-mannosidase I inhibitor 1-deoxymannorjirimycin. Transport between the ER and Golgi complex in vitro requires the addition of a high speed supematant (cytosol) of cell homogenates, and requires energy in the form of ATP. Efficient reconstitution of export of protein from the ER requires the preparation of homogenates from mitotic cell populations in which the nuclear envelope, ER, and Golgi compartments have been physiologically disassembled before cell homogenization. These results suggest that the high efficiency of transport observed here may require reassembly of functional organelles in vitro. A understanding of the biochemical mechanisms that control the vesicular transport of protein between compartments of the secretory pathway of eucaryotic cells will require successful reconstitution of individual segments of the pathway using novel cell-free systems. Transport of membrane glycoprotein between the cis and medial compartments of the Golgi complex has been successfully reconstituted in vitro (3, 13, 21, 22). Studies in this system are providing, for the first time, new insight into the biochemical mechanisms involved in intercompartmental transfer in this organelle (5, 49, 73, 74). Other stages in the exocytic pathway remain to be reconstituted in vitro with high efficiency. An important step in the delivery of protein to the cell surface is the selective export of proteins from their site of synthesis in the cellular endoplasmic reticulum (ER). I The biochemical basis for synthesis (72), insertion (75), and core-glycosylation (55) of protein in the rough endoplasmic reticulum (RER) has been the subject of intense investigation. In contrast, little is known of the subsequent biochemical events that regulate export of protein from the ER to the next compartment of the exocytic pathway, the Golgi complex. 1. Abbreviations used in this paper: CP, creatine phosphate; CPK, rabbit muscle creatine phosphokinase; DMM, 1-deoxymannojirimycin; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; RER, rough endoplasmic reticulum. Isolation of yeast temperature-sensitive mutants that are pleiotropically defective in export of both membrane-bound and secreted protein from the ER has established that a common biochemical basis regulates trafficking from this organelle (48). Comparative studies defining differences in the kinetics of transport of a variety of membrane-bound and secreted proteins (23, 40, 61, 65, 76), the altered secretion of genetically engineered mutant proteins (19, 24, 56, 57), and the requirement for cofactors (53) or quaternary interactions (36, 37, 46, 50, 64) for export, suggest that signals encoded in the primary sequence of a protein provide important information, facilitating an initial step in interaction with this common transport machinery. Transport is vesicular; proteins are delivered between compartments through the budding and specific fusion of carrier vesicles (51). The enzymological basis for these events in the exocytic pathway remains to be defined. To begin to understand the first stage of transport, export of newly synthesized protein from the ER, we are using a model system based on the G protein of vesicular stomatitis virus (VSV). The synthesis (31, 41), posttranslational processing (34, 35, 42, 66, 68), and transport of G protein from the ER to the cell surface (7, 8) has been extensively studied in vivo, and is indistinguishable from normal cellular membrane-bound glycoproteins, providing a sound experimental © The Rockefeller University Press, 0021-9525/87103/749/12 $1.00 The Journal of Cell Biology, Volume 104, March 1987 749-760 749 on N ovem er 6, 2017 jcb.rress. org D ow nladed fom system to explore new avenues of understanding of the secretory pathway. During synthesis, G protein acquires two high mannose core-oligosaccharides (42). Transfer of G protein from the ER to the cis compartment of the Golgi in vivo results in the G protein oligosaccharides being trimmed from the mans_9GlcNAc2 forms to the mansGlcNAc2 species as a result of the early Golgi oligosaccharide-processing enzyme ¢t-mannosidase I (1, 4, 34, 35, 67). Trimming of G protein can be followed directly in the mutant Chinese hamster ovary (CHO) cell line clone 15B, which is missing the medial Golgi processing enzyme N-acetylglucosamine transferase I (16, 26). As a consequence, further processing of the mansGlcNAc2 oligosaccharide form to the complex structure by sequentially acting ct-mannosidase II and glycosyltransferases in later compartments of the Golgi is blocked (15, 18, 34). G protein is delivered to the cell surface in the mansGlcNAc2 form, since processing, not transport, is the sole defect in clone 15B (62). Export of G protein from the ER in vivo can be readily distinguished from later transport steps between Golgi compartrnents through use of the temperature-sensitive VSV mutant strain ts045, in which G protein is found exclusively in the ER when ceils are incubated at the restrictive temperature (39.5°C) during infection (32, 33, 39, 44, 63, 77). Shift of cells from the restrictive to the permissive temperature (32°C) in vivo results in the synchronous release of G protein to the Golgi complex as shown both morphologically (7, 8) and biochemically through trimming to the mansGlcNAc2 form (4). Transport of ts045 G protein between the ER and the Golgi apparatus in vivo has been shown to occur in two biochemically distinct steps: an early temperature-sensitive, ATP-dependent export (budding) step, followed by a temperatureand ATP-insensitive step providing for delivery (fusion) of G protein to the cis Golgi compartment containing ¢t-mannosidase I (1, 4). In this paper, using the transport of G protein as a model system, we provide evidence that we have successfully reconstituted a new stage of the exocytic pathway in vitro, transport of protein from the ER to a cis Golgi compartment containing a-mannosidase I. Transport is highly efficient and proceeds with kinetics similar to those observed in vivo. Transport occurs between sealed membrane-bound compartments, requires energy in the form of ATP, and is dependent upon the addition of a cytosol fraction containing soluble components prepared from a high speed supernatant of centrifuged cell homogenates. Materials and Methods