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

The Open Biochemistry Journal 28 Apr 2017 RESEARCH ARTICLE DOI: 10.2174/1874091X01711010036



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.


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 ubiquitylated


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 COPII


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.


Our results suggest a novel regulatory mechanism for the Sec23a function that could be crucial in several pathophysiological events known to alter COPII recycling

Keywords: Ubiquitylation, Sec23a, COPII, ERES, Vescicular transport.
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