Randy Schekman, investigator at the Howard Hughes Medical Institute and professor of molecular and cell biology at the University of California, Berkeley, presented a seminar last Tuesday on the current status of membrane trafficking research and its application to human diseases.
Knowledge on the mechanism of membrane trafficking has greatly advanced since the 1970s after Schekman’s lab, which studied the secretory pathways (cellular secretion of proteins) using genetically manipulated yeast cells, identified a multitude of genes involved in vesicle transport. Such genetic screenings were subsequently accompanied by not only the investigation of the functional roles that the identified proteins play, but also the categorization of several human diseases that may arise from defects in the secretory pathway.
Over thirty years later, our knowledge of membrane trafficking has expanded generously, but the details of the mechanism continue to be as mysterious and intriguing. One peculiar aspect of membrane trafficking is how the SEC13/31 COPII coat cage manages to contain material seemingly larger than the cage itself. The COPII coat consists of SAR1 GTPase, SEC23-24 adaptors, and an outer layer of SEC13-31 heterotetramers.
COPII vesicles play a critical role in the transport of proteins from the rough Endoplasmic Reticulum (rER) to the Golgi apparatus. Given the extensive amount of protein trafficking from one cell compartment to another, vesicle transport would all but breakdown without a means of regulating the formation of transport vesicles and selectively storing proteins into those vesicles. The COPII coating provides structure and selectivity to biogenesis of transport vesicles. Once a region of the rER membrane is ready to be pinched off as a vesicle, Sar1 recruits the heterotetramer SEC13/31, which forms the shape of a cage and provides the COPII vesicle its structural integrity.
Failure of the COPII function leads to many potential issues, including collagen-deposition defects, cranio-lenticulo-sutural dysplasia, or chylomicron retention disease. Some of these diseases reflect the critical role that COPII vesicles play in transporting procollagen, a role that is complicated by the fact that COPII vesicles (60-80nm diameter) are usually much smaller than the 300-400nm procollagen fibers.
The mechanism by which COPII vesicles expand to accommodate the much larger procollagen fibers remained an enigma until very recently, when Schekman and Rape identified the ubiquitin ligase CUL3-KLHL12 as the regulator of COPII coat formation by means of ubiquitylation (the addition of ubiquitin to the target protein). An siRNA screen in mouse embryonic stem cells revealed that knockdown of CUL3 led to significant alteration of the cells’ structural morphology, a defect which was rescued by addition of collagen. This suggested to the researchers a potential link between collagen secretion and CUL3 (as well as KLHL12, which interacts with CUL3 for ubiquitylation). Immunoprecipitation showed that SEC31 but not SEC13 was found to strongly co-immunoprecipitate with KLHL12, suggesting that CUL3-KLHL12 influences the COPII coat by monoubiquitylating SEC31. Immunocytochemistry additionally show colocalization of KLHL12 with COPII, further supporting CUL3-KLHL12’s involvement. The researchers speculated that monoubiquitylation of SEC31 very likely caused a conformational change that greatly increased the size of the COPII coat cage created by the SEC13/31 heterotetramer. Confocal microscopy revealed that KLHL12-transfected IMR90 cells formed large, hollow, spherical structures (200-500nm) suggestive of expanded COPII vesicles. Exogenous expression of KLHL12 also greatly improved the efficiency of collagen transport in IMR90 cells, showing the necessity of CUL3-KLHL12 for collagen export.
These findings not only have therapeutic significance in the development of new strategies to combat known diseases, but also provide new insight into membrane trafficking. It remains a mystery how ubiquitylation actually increases COPII coat size or structure; resolving this mystery would be one of the next steps for improving our understanding of membrane trafficking.