Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER

Abstract

Background:COPII is a multiprotein complex that surrounds carrier vesicles budding from the Endoplasmic Reticulum and allows the recruitment of secretory proteins. The Sec23a protein plays a crucial role in the regulation of the dynamics of COPII formation ensuring the proper function of the secretory pathway.Objective:Since few evidences suggest that ubiquitylation could have a role in the COPII regulation, the present study was aimed to establish whether the Sec23a component of the vesicular envelope COPII could be ubiquitylatedMethod:Sec23a ubiquitylation was revealed by co-immunoprecipitation experiments. Recombinant Sec23a was gel-purified and analyzed by mass spectrometry subjected to trypsin proteolysis. Signature peptides were identified by the presence of Gly–Gly remnants from the C-terminus of the ubiquitin attached to the amino acid residues of the substrate. Recombinant Sec23a proteins bearing mutations in the ubiquitylation sites were used to evaluate the effect of ubiquitylation in the formation of COPIIResults:We identified two cysteine ubiquitylation sites showed at position 432 and 449 of the Sec23a protein sequence. Interestingly, we revealed that the amino acid residues of Sec23a joined to ubiquitin were cysteine instead of the conventional lysine residues. This unconventional ubiquitylation consists of the addition of one single ubiquitin moiety that is not required for Sec23a degradation. Immunofluorescence results showed that Sec23a ubiquitylation might influence COPII formation by modulating Sec23a interaction with the ER membrane. Presumably, this regulation could occur throughout continual ubiquitylation/de-ubiquityliation cycles.Conclusion:Our results suggest a novel regulatory mechanism for the Sec23a function that could be crucial in several pathophysiological events known to alter COPII recycling

Export Control: Post-transcriptional Regulation of the COPII Trafficking Pathway

“…Ubiquitination may also regulate mammalian Sec23. Recently, the Remondelli group reported the unusual monoubiquitination of human Sec23A on two cysteine residues, C432 and C449 ( Amodio et al, 2017 ). These residues may be functionally important for COPII trafficking, as the authors report that a C→A mutation at either site reduces Sec23A occupancy at ERES in cultured mammalian cells, though the mechanisms and consequences of these proposed PTMs remain to be confirmed in trafficking assays ( Amodio et al, 2017 ).…”

The Main Determinants of Diabetes Mellitus Vascular Complications: Endothelial Dysfunction and Platelet Hyperaggregation

“…It has been demonstrated that circulating total microparticles are greatly elevated in patients with T2DM, suggesting that changing microparticle levels are possibly relevant to diabetic conditions [ 43 ], thus opening a new scenario for future investigations. Recently, it has also been demonstrated that Sterol regulatory element-binding protein 1 (SREBP1)—an important transcriptional regulator of lipogenesis [ 44 ], regulated by Sec23A [ 45 ]—is involved in the modulation of insulin resistance and in the lipogenesis that is chronically enhanced diabetes, thus laying the foundation for the development of novel future therapeutic strategies to contain the onset of diabetes.…”

ER exit pathways and the control of proteostasis: Crucial role of the UPR, COPII, and ER-phagy in the secretory pathway

“…Indeed, in driving vesicle formation, COPII ensures by Sec24 isotypes that only folded transmembrane proteins or soluble cargo molecules are sorted and packaged by specific cargo receptors, respectively. The flux of COPII vesicle carriers exiting the ER is regulated by different factors according to secretory demand (Subramanian et al, 2019) or to the ER stress conditions, in which the abundance of COPII vesicles is reduced, giving rise to alterations in cargo secretion and structural defects of post-ER compartments (Amodio et al, 2009;Amodio et al, 2013;Amodio et al, 2017).…”