KLHL12 can form large COPII structures in the absence of CUL3 neddylation

Abstract

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.

Dynamic regulation of inter-organelle communication by ubiquitylation controls skeletal muscle development and disease onset

“…While most procollagens are trafficked through the secretory pathway, a subset is directed towards lysosomal degradation to remove excess procollagen from cells through the autophagy pathway ( Omari et al, 2018 ). Recent studies in skin fibroblasts have shown that the CUL3-KLHL12 complex is involved in routing procollagens to lysosomes to regulate intracellular collagen levels ( Moretti et al, 2023 ). Moreover, inhibition of CUL3 neddylation which is critical for the ubiquitylation activity still led to the formation of large COPII vesicles by the CUL3-KLHL12 complex, which is required for the secretion of procollagens.…”

Steady-state regulation of COPII-dependent secretory cargo sorting by inositol trisphosphate receptors, calcium, and penta EF hand proteins

“…However, neither ALG-2 nor peflin are required for secretory trafficking and peflin depletion increases collagen ER-to-Golgi transport in NRK cells ( 8 ). Furthermore, inhibition of CUL3 ubiquitylation activity with neddylation inhibitors did not affect collagen secretion, but instead CUL3 KLHL12 appeared to regulate ER-lysosome trafficking for degradation ( 47 ). This also fits with recent observations that COPII and KLHL12 colocalized with procollagen at sites of noncanonical ER-phagy ( 48 ).…”

TANGO1 Dances to Export of Procollagen from the Endoplasmic Reticulum

“…This monoubiquination delays the closure of the vesicle, allowing for more procollagen to be incorporated. Studies have also shown the neddylation of CUL3 to be important in the process of delaying vesicle closure [ 57 ], whereas others have shown it not to be important [ 58 ]. The mild over-expression of KLHL12 leads to the formation of large COPII vesicles [ 56 ], suggesting a direct role for KLHL12/CUL3 in procollagen export.…”