Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes

Abstract

We have previously shown that influenza haemagglutinin (HA) acquires Endo H resistance en route to the cell surface after microinjection of its mRNA into Xenopus oocytes (Ceriotti, A. and A. Colman. 1989. J. Cell Biol. 109:1439-1444.) In this paper we use the injection of varying amounts of mRNA (0.05-5 ng/oocyte) to effect a 30-fold change in HA protein synthesis within the oocyte. Using the Endo H assay as an indicator of protein movement from the ER to the medial Golgi we find that this movement is reduced, sometimes dramatically, when intracellular HA levels fall. This reduction in movement is closely correlated with a decreased rate of trimer formation as assessed both by trypsin resistance and sedimentation analysis, leading us to conclude that trimer formation is not only, as has been shown before essential for ER-Golgi complex movement, but is the major rate limiting step in this movement. Interestingly at least 50% of unassembled HA monomers that accumulate after low HA synthesis can be rescued into trimers over 24 h later, after a second injection of concentrated HA mRNA. In contrast when we repeated this experiment with another membrane protein, the human low density lipoprotein, or with murine secretory immunoglobulin we found that the rate of movement was insensitive to the protein concentration. This latter result seemed surprising since earlier work had shown that unassembled IgG heavy chains (like monomeric HA) remain in the oocyte ER; however in these present experiments we have been unable to detect any unassembled heavy chains even at the lowest expression levels, indicating that tetramerization of Ig is much faster than trimerization of HA.