Targeting of protein ERGIC-53 to the ER/ERGIC/cis-Golgi recycling pathway

Since endogenous ERGIC-53 has not been found in compartments beyond the cis-Golgi we have exploited the acquisition of endo H resistance of the glycosylated ERGIC-53 variant GM as a principal measure for the rate of loss from the ER-ERGIC-cis-Golgi system. This assay reveals subtle changes in the overall retention of GM mutants in the pre-mediaI-Golgi recycling pathway, provided the expression levels of the different constructs are comparable, a condition that was met in our experiments. However, the assay does not discriminate between recycling of overexpressed ERGIC-53 (GM) from post-medial-Golgi compartments and transport to the cell surface. Three findings argue against a major contribution of recycling from late Golgi sites. First, ER-localized GM in highly overexpressing COS cells failed to costain with FITC-ricin, a lectin recognizing -(1,4)-linked galactose added in the trans-Golgi (not shown). Jackson et al. (1993) reported ER staining by Helix pomatia lectin (recognizing terminal GalNAc) and peanut lectin (recognizing terminal Gal-(1,3)-GalNAc) in cells overexpressing an O-glycosylated CD8/E19 chimeric protein possessing a di-lysine ERretrieval signal. While this result is evidence for recycling of a di-lysine protein via the Golgi apparatus it does not necessarily indicate recycling from the trans-Golgi. Helix pomatia lectin recognizes terminal GalNAc added both in the cis-Golgi (Roth et al., 1994) and the trans-Golgi (Roth, 1984) , and peanut lectin binds glycans in the medial-Golgi (Roth, 1987) . Second, we found a strict correlation between the acquisition of endo H resistance and mislocalization to the cell surface of the different mutant proteins. Third, we have shown that ERGIC-53 contains a functional di-lysine ER-retrieval signal, and a recycling pathway from the ERGIC and the cis-Golgi is consistent with the major localization of coatomer, the putative receptor for di-lysine signals (Oprins et al., 1993; Cosson and Letourneur, 1994; Letourneur et al., 1994) . Collectively, our data argue against ERGIC-53 recycling from the trans-Golgi to biosynthetically earlier compartments. We conclude therefore that the acquisition of endo H resistance reflects loss of intracellular retention of ERGIC-53.