Silkworm posterior silkgland is a model for studying intracellular trafficking. Here, using this model, we identify several potential cargo proteins of BmKinesin-1 and focus on one candidate, BmCREC. BmCREC (also known as Bombyx mori DNA supercoiling factor, BmSCF) was previously proposed to supercoil DNA in the nucleus. However, we show here that BmCREC is localized in the ER lumen. Its C-terminal tetrapeptide HDEF is recognized by the KDEL receptor, and subsequently it is retrogradely transported by coat protein I (COPI) vesicles to the ER. Lacking the HDEF tetrapeptide of BmCREC or knocking down COPI subunits results in decreased ER retention and simultaneously increased secretion of BmCREC. Furthermore, we find that BmCREC knockdown markedly disrupts the morphology of the ER and Golgi apparatus and leads to a defect of posterior silkgland tube expansion. Together, our results clarify the ER retention mechanism of BmCREC and reveal that BmCREC is indispensable for maintaining ER/Golgi morphology.
Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgi-to-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation.
Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20-46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2.
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