SEC24 is mainly involved in cargo sorting during COPII vesicle assembly. There are four SEC24 paralogs (A-D) in vertebrates, which are classified into two subgroups (SEC24A/B and SEC24C/D). Pathological mutations in SEC24D cause osteogenesis imperfecta with craniofacial dysplasia in humans. sec24d mutant fish also recapitulate the phenotypes. Consistent with the skeletal phenotypes, the secretion of collagen was severely defective in mutant fish, emphasizing the importance of SEC24D in collagen secretion. However, SEC24D patient-derived fibroblasts show only a mild secretion phenotype, suggesting tissue-specificity in the secretion process. Using Sec24d KO mice and cultured cells, we show that SEC24A and SEC24B also contribute to endoplasmic reticulum (ER) export of procollagen. In contrast, fibronectin 1 requires either SEC24C or SEC24D for ER export. On the basis of our results, we propose that procollagen interacts with multiple SEC24 paralogs for efficient export from the ER, and that this is the basis for tissue-specific phenotypes resulting from SEC24 paralog deficiency.

CUL3-RING ubiquitin ligases (CRL3s) are involved in diverse cellular processes through over two hundred BTB-domain proteins. KLHL12, a BTB-domain protein, has been suggested to play an essential role in export of unusually large cargo molecules like procollagen from the endoplasmic reticulum (ER). It has been suggested that CRL3 KLHL12 mono-ubiquitinates SEC31 and mono-ubiquitinated SEC31 increases the dimension of a COPII coat to accommodate the large cargo molecules. As we examined this model, we found that functional CRL3 KLHL12 was indeed critical for the assembly of large COPII structures. However, it did not directly affect collagen secretion, but instead influenced collagen synthesis in human skin fibroblasts (HSFs).These results also suggest that there is a CRL3 KLHL12 -independent collagen secretion route. Unexpectedly, CRL3 KLHL12 strongly influenced the levels of sensors of the unfolded protein response (UPR) such as PERK and IRE1α. Interestingly, different cell lines reacted differently to CUL3 depletion. This cell-line dependency appears to rely on a cell-line specific BTB-domain protein(s) and a cell-line specific substrate(s) of the BTB-domain protein. Consistent with this idea, depletion of a muscle-specific BTB-domain protein KLHL41 recapitulated the effects of CUL3 depletion in C2C12 myotubes. Based on these results we propose that CRL3 KLHL12 and CRL3 KLHL41 are regulators of the UPR sensors. expression levels.

CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric a brac, Tramtrack and Broad-Complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, is suggested to play an essential role in the export of large cargo molecules like procollagen from the endoplasmic reticulum (ER). CRL3KLHL12 mono-ubiquitylates SEC31, leading to an increase in COPII vesicle dimension. Enlarged COPII vesicles can accommodate procollagen molecules. Thus, CRL3KLHL12 is essential for the assembly of large COPII structures and collagen secretion. CRL3s are activated by CUL3 neddylation. Here, we evaluated the importance of CUL3 neddylation in COPII assembly and collagen secretion. Unexpectedly, the assembly of large COPII-KLHL12 structures persisted and cellular collagen levels decreased by the treatment of MLN4924, a potent inhibitor of NEDD8-activating enzyme. When we introduced mutations to KLHL12 at the CUL3 interface, these KLHL12 variants did not interact with neddylated CUL3, but one (Mut A) of them still supported large COPII-KLHL12 structures. Overexpression of wild-type KLHL12, but not Mut A, lowered cellular collagen levels most likely via lysosomal degradation. Our results suggest that CUL3 neddylation is not necessary for the formation of large COPII-KLHL12 structures, but active CRL3KLHL12 contributes to the maintenance of collagen levels in the cell.