Date Published: April 13, 2012
Publisher: Public Library of Science
Author(s): Qiao Wang, Hui Feng, Pengli Zheng, Birong Shen, Liang Chen, Lin Liu, Xiao Liu, Qingsong Hao, Shunchang Wang, Jianguo Chen, Junlin Teng, Sara Salinas. http://doi.org/10.1371/journal.pone.0035344
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.
Human calumenin (calu), a CREC protein family member, is encoded by the CALU gene (NCBI GeneID: 813) , , which is mapped on chromosome 7q32 . Two alternative spliced variants of the CALU gene are identified as calu-1 and calu-2 (also known as crocalbin) . The two isoforms have equal lengths (315 amino acids), with exons 3 and 4 exchanged , and are ubiquitously expressed in human tissues . Both calu-1 and -2 contain an N-terminal signal sequence (19 amino acids) and seven EF-hand domains for binding Ca2+. Previous reports show that they localize to the secretory pathway and are secreted into the extracellular space –, while some researches insist that calu-1/2 contained an ER-retaining signal HDEF at the C-terminus, and are retained in the ER lumen –. Besides, proline at the +2 position from the predicted signal peptide cleavage site of calu-1/2  acts as an export signal to mediate calu-1/2 secretion .
In this study, we showed that calu-1/2-EGFP are translocated into the lumen with the help of their signal peptide, transported intracellularly in vesicles, and finally secreted into the extracellular space (Figure 9), during which the sorting signal on calu-1/2 is indispensable. We examined the velocities of calu-1/2-EGFP-containing vesicles, and identified the motor proteins that participate in powering calu-1/2-EGFP-containing vesicles. Most interestingly, besides the traditional mode of secretion, we also observed another type of secretion, in which calu-1/2-EGFP-containing vesicles accumulated in cellular processes and were secreted collectively.