Polymerization of Calsequestrin Inside the Secretory Pathway is Isoform-Specific and Occurs on Either Side of ER Exit Sites

Abstract

In heart and fast twitch skeletal muscle, cardCSQ and skelCSQ concentrate in an ER/SR compartment known as junctional SR. Junctional SR is morphologically distinct in the two cell types, and mechanisms of CSQ trafficking and concentration within junctional SR remain undetermined. A model for CSQ polymerization has recently been developed that could explain traditional observations of a matrix of CSQ inside junctional SR lumens. CardCSQ, for example, is very efficiently retained in proximal ER tubules, as long as its native structure is not critically modified, or the ER milieu significantly altered. In the present study, we found that skelCSQ trafficked differently from cardCSQ in nonmuscle cells and neonatal heart cells. In fact, the distribution of skelCSQ was unique among known ER markers. SkelCSQ concentrated in a membrane compartment that was juxtaposed and distal to ER containing the cardiac isoform. SkelCSQ was contained in novel streaming tubules and vesicles aligned on tufts of microtubules. Consistent with immunofluorescence microscopy were the structures of the two Asn316-linked glycans on CSQ isoforms, with skelCSQ glycans trimmed beyond the Man9,8 that are indicative of proximal ER. Despite the complete non-overlap of skelCSQ and cardCSQ compartments, the two proteins co-localized in early ER when co-overexpressed, suggesting heteropolymer formation. The present study indicates that skelCSQ, in contrast to cardCSQ, evades ER polymerization, and polymerizes in the next distal secretory compartment, an early subcompartment of ERGIC. We conclude that different subcellular localizations for skelCSQ and cardCSQ result from a difference in the lumenal requirements for polymerization of each of the two CSQ isoforms, leading to ER retention (cardCSQ) or retention in a contiguous intermediate compartment (skelCSQ).