Subcellular localization of ERGIC-53 under endoplasmic reticulum stress condition

Newly synthesized glycoproteins destined for secretion aretransported from the endoplasmic reticulum (ER),through the Golgi and toward the cell surface. In this se-cretion pathway, several intracellular ER- or Golgi-resi-dent transmembrane proteins serve as cargo receptors.ER–Golgi intermediate compartment (ERGIC)-53, VIP36and VIPL, which have an L-type lectin domain within theluminal portion, participate in the vectorial transport ofglycoproteins via sugar–protein interactions. To under-stand the nature of these receptors, monoclonal antibodieswere generated against human ERGIC-53, VIP36 andVIPL using 293T cells expressing these receptors on cellsurfaces. These cells were used to immunize rats and forscreening antibody-producing clones. Flow cytometricanalysis and immunoprecipitation studies showed that theobtained monoclonal antibodies bound specifically to thecorresponding cargo receptors. Immunostaining of HeLacells using the monoclonal antibodies showed that the lo-calization of ERGIC-53 changed from relatively broad dis-tribution in both the ER and the Golgi under normalconditions to a compact distribution in the Golgi underER stress conditions. This redistribution was also observedby the overexpression of ERGIC-53 and abrogated by co-expression with VIPL but not VIP36. Real-time polymer-ase chain reaction revealed that ERGIC-53 along withseveral chaperone proteins was up-regulated after tunica-mycin treatment; however, the expression of VIPL was un-changed. Furthermore, ERGIC-53 co-precipitated withVIPL but not VIP36, indicating that ERGIC-53 may inter-act with VIPL in the ER, which may regulate the localiza-tion of ERGIC-53 inside cells. Taken together, theseobservations provide new insights into the regulation ofthese cargo receptors and the quality control of glycopro-teins within cells.Keywords: cargo receptor/ERGIC-53/ER stress/glycoprotein quality control/VIPLIntroductionIn many cases, glycosylation directly affects the properties ofproteins and cells, sometimes with important biological conse-quences. The biological functions of glycosylation are mostlymediated outside the cell (Ashwell and Morell 1974; Crockerand Varki 2001; Ley 2003). In contrast, oligosaccharides, es-pecially N-glycans, function as tags for the quality control ofglycoproteins within cells. Recently, the biological mechan-isms regulating folding, transport and endoplasmic reticulum(ER)-associated degradation of glycoproteins have becomebetter understood (Helenius and Aebi 2004; Moremen andMolinari 2006; Ruddock and Molinari 2006; Yamamoto2009). The extension of protein folding, or the onset of dis-posal, is regulated by several ER- and Golgi-resident sugar-binding proteins (called lectins), which recognize specificN-glycan structures attached to proteins. We recently deter-mined the sugar-binding specificity of the intracellular cargoreceptors, ER–Golgi intermediate compartment (ERGIC)-53,VIP36 and VIPL, using a flow cytometry-based method in-corporating soluble lectin tetramers (Kawasaki et al. 2007,2008; Yamaguchi et al. 2007; Yamamoto and Kawasaki 2010)and frontal affinity chromatography (Kamiya et al. 2005,2008). We found that progressive trimming of terminal sugarresidues is required to recruit cargo receptors. However, theintracellular localization of these receptors is necessary if weare to understand their biological roles, particularly withrespect to the transport of cargo proteins. The cargo receptors,ERGIC-53, VIP36 and VIPL, are homologous at the aminoacid level; however, monoclonal antibodies that distinguishbetween these cargo receptors have not yet been reported.VIP36 localization to either the pre-Golgi secretory pathwayor the post-Golgi pathway was shown using endogenouslyexpressed Myc-tagged or FLAG-tagged receptors and anti-tagantibodies (Fiedler et al. 1994; Fullekrug et al. 1999 ), and arecent report identified VIP36 mainly on the cell surface(Shirakabe et al. 2011). ERGIC-53, which was first identified asa marker of the ERGIC, is distributed between the ER and theGolgi (Vollenweider et al. 1998; Appenzeller et al. 1999),whereas VIPL localized predominantly in the ER and may be anon-cycling ER-resident protein ( Nufer, Mitrovic, et al. 2003);however, there may be the possibility that exogenouslyexpressed tagged protein does not co-localized with endogenous