|dc.description.abstract||The COPII machinery is the primary mediator of ER-to-Golgi transport and mutations in different COPII (Coat Protein II complex) paralogs are associated with diverse human diseases. Each tissue has vastly different requirements for the secretory machinery in terms of cargo type and volume, and COPII must accommodate these requirements. The mechanisms by which COPII function may be regulated, however, are only beginning to be elucidated. Because skeletal tissues are highly secretory and traffic large cargoes such as collagen, we used forward genetics approaches in zebrafish coupled with a positional cloning strategy to identify novel genes important for protein trafficking in skeletal tissues. Here we identify a mutation in the transcription factor feelgood/creb3l2, which directly regulates the expression of genes encoding select COPII proteins, including sec23a and sec24d. We found that these COPII components, but not their close paralogs sec23b or sec24c, are required for collagen secretion in chondrocytes and the notochord, but not for secretion of other cargos such as peptidoglycans, laminin, and N-cadherin.
Loss of function of sec23a and sec23b paralogs leads to highly divergent phenotypes. We found that collagen deposition in the notochord is dependent on Sec23a while fibronectin deposition is dependent on Sec23b. To evaluate whether the unique phenotypes are due to differences in expression or function of sec23 paralogs, we have performed rescue experiments in crusher/sec23a mutants. sec23a overexpression in crusher was able to rescue morphology of the pectoral fin, and collagen trafficking within the fin, significantly better than sec23b overexpression. These data suggest a functional difference between the paralogs. Because Sec23a and Sec23b amino acid sequence is 93% similar except for a highly divergent 18 amino acid loop, we hypothesized that this loop may be essential for their functional difference. We therefore build a sec23b construct containing the 18AA loop from sec23a, as well as the reciprocal sec23a with the sec23b loop. In contrast to WT sec23b, overexpression of sec23b with the sec23a loop was able to efficiently rescue collagen secretion and fin morphology in crusher/sec23a mutants, while sec23a with the sec23b loop failed to do so. These data indicate that functional differences between sec23a and sec23b exist and that the highly divergent 18 amino acid loop plays a role in collagen secretion.
Our work has established that Creb3l2/Sec23a/Sec24D are critical for collagen/ECM secretion during the maturation stage of chondrogenesis and suggests that this secretory axis is central to a broad transcriptional regulatory network that optimizes cellular machinery to facilitate trafficking in highly secretory cell types.||