Formation and IntraceUular Transport of a Heterodimeric Viral Spike Protein Complex

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a ll0,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER. S ECRETORY and membrane proteins are synthesized and acquire asparagine-linked glycans in the ER. In most cases, they then embark onto the exocytic transport pathway to the plasma membrane (PM) ~ via the Golgi complex (GC) (17, 23, 24, 42). Our understanding of the early steps involved in this exocytic pathway, i.e., disulfide bond formation, folding and oligomerization of membrane proteins has improved substantially during the last few years. It now appears clear that an elaborate quality control of protein folding and oligomerization exists in the ER that prevents unor misfolded and unassembled proteins from leaving this compartment (19, 24, 37, 44, 46). The maturation process may involve the catalytic action of several proteins such as the IgG heavy chain binding protein (BiP or GRP78) (37, 46), protein disulfide isomerase (PDI) (11, 12), glycosylation site binding protein (15), and perhaps yet other chaperoninlike proteins. It has been known for a long time that various secretory and membrane proteins have very different transport kinetics from the ER to the GC (10, 13, 30, 34). An apparent possibility is that protein folding and oligomerization is the rate-limiting step. In agreement with this, it has been found that different proteins fold and assemble into oligomers with vastly different kinetics. Oligomerization seems to 1. Abbreviations used in this paper: BiP, IgG heavy chain binding protein; GC, Oolgi complex; PDI, protein disulfide isomerase; PM, plasma membrane. be an important prerequisite for exit of membrane proteins from the ER compartment (19, 44). Studies on the folding and oligomerization of cellular proteins, such as procollagens (22), the T cell receptor (24, 33), glycoprotein hormones (48), class I and class II MHC antigens, and immunoglobulins (for references, see 19), have revealed the intricate steps involved in protein folding and oligomerization in the ER. Many of the details involved in the early steps have been elucidated using two viral model systems, the hemagglutinin of influenza virus (2, 5, 6, 16) and the G protein of vesicular stomatitis virus (VSV) (7, 8, 25, 31). These model proteins have many features in common, such as the kinetics of folding, disulfide bond formation, and trimerization, which in both cases take ~7-10 min (6-8, 16). Properly folded and trimerized proteins are then rapidly transported to the GC. Misfolded proteins, on the other hand, tend to aggregate and are retained in the ER (19). In only a few cases has it been directly shown that proteins are transiently associated with BiP during the folding process (1, 9, 31, 35, 47). Although the exact role of BiP is still under debate, it may assist in the folding process, prevent aggregation and prevent unor misfolded proteins from exiting the ER (18, 19, 37). We are using a different viral model system, the glycoproteins of bunyaviruses and in particular those of Uukuniemi virus, to study the above processes. AI1 bunyaviruses contain two glycoproteins, G1 and G2, tha! form the surface projec© The Rockefeller University Press, 0021-9525/91/01/257/10 $2.00 The Journal of Cell Biology, Volume 112, Number 2, January 1991 257-266 257 on May 3, 2019 jcb.rupress. org Downloaded from http://doi.org/10.1083/jcb.112. 2.257 Published Online: 15 January, 1991 | Supp Info: