CS (calsequestrin) is an acidic glycoprotein of the SR (sarcoplasmic reticulum) lumen and plays a crucial role in the storage of Ca2+ and in excitation–contraction coupling of skeletal muscles. CS is synthesized in the ER (endoplasmic reticulum) and is targeted to the TC (terminal cisternae) of SR via mechanisms still largely unknown, but probably involving vesicle transport through the Golgi complex. In the present study, two mutant forms of Sar1 and ARF1 (ADP-ribosylation factor 1) were used to disrupt cargo exit from ER-exit sites and intra-Golgi trafficking in skeletal-muscle fibres respectively. Co-expression of Sar1-H79G (His79→Gly) and recombinant, epitope-tagged CS, CSHA1 (where HA1 stands for nine-amino-acid epitope of the viral haemagglutinin 1), barred segregation of CSHA1 to TC. On the other hand, expression of ARF1-N126I altered the subcellular localization of GM130, a cis-medial Golgi protein in skeletal-muscle fibres and myotubes, without interfering with CSHA1 targeting to either TC or developing SR. Thus active budding from ER-exit sites appears to be involved in CS targeting and routing, but these processes are insensitive to modification of intracellular vesicle trafficking and Golgi complex disruption caused by the mutant ARF1-N126I. It also appears that CS routing from ER to SR does not involve classical secretory pathways through ER–Golgi intermediate compartments, cis-medial Golgi and trans-Golgi network.
Abbreviations used: AP, adaptor protein; ARF1, ADP-ribosylation factor 1; BFA, Brefeldin A; BiP, immunoglobulin heavy-chain binding protein; COPI, coat protein complex I; CS, calsequestrin; DMEM, Dulbecco's modified Eagle's medium; ER, endoplasmic reticulum; ERGIC, ER–Golgi intermediate compartment; GC, Golgi complex; GFP, green fluorescent protein; HA1, nine-amino-acid epitope of the viral haemagglutinin 1; PDI, protein disulphide isomerase; RYR, ryanodine receptor; SR, sarcoplasmic reticulum; TC, terminal cisternae; TGN, trans-Golgi network; VSV-G, vesicular-stomatitis-virus glycoprotein.