Protein Recycling from the Golgi Apparatus to the Endoplasmic Reticulum in Plants and Its Minor Contribution to Calreticulin Retention

Abstract

Using pulse-chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post-endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan-related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs. INTRODUCTIONIn eukaryotic cells, most proteins that reside in the endoplasmic reticulum (ER) contain information in their primary structure that determines their subcellular localization. Keeping these proteins in the ER can be achieved either by strict retention in this particular compartment or by retrieval and retrograde transport back to the ER when they leave the ER (reviewed in Gomord et al., 1999;Pagny et al., 1999; Vitale and Denecke, 1999). For soluble ER resident proteins or soluble reticuloplasmins, ER residency depends largely on the presence of the specific tetrapeptide H/KDEL-or closely related sequences-at their C termini (Munro and Pelham, 1987;Andres et al., 1990). Use of different tetrapeptide sequences fused to non-ER secretory proteins as reporter probes has shown that these signals are sufficient for retention of soluble proteins in the ER (Rose and Doms, 1988;Herman et al., 1990; Denecke et al., 1992; Wandelt et al., 1992; Boevink et al., 1996;Gomord et al., 1997). Moreover, for yeast and mammalian cells, the H/KDEL-dependent retention mechanism has been proposed to promote a continual recycling of ER resident proteins whenever they exit the ER and are secreted into a post-ER compartment (Pelham, 1988).Arguing for such a recycling of ER proteins is the observation that reporter glycoproteins fused with H/KDEL motifs undergo specific N-glycan modifications. Indeed, the latter are markers of post-ER events. For example, in animal cells, the lysosomal glycoprotein cathepsin D, when modified by the addition of a KDEL C-terminal extension, accumulates in the ER even though it carries N-glycans containing the N -acetylglucosamine 1-phosphate residues that are typical of passage through the cis Golgi compartment (Pelham, 1988). Some post-ER glycan maturations have also been found on reporter glycoproteins ...

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