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 ubiquitylated.

Method: 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.

Results: 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.

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

Fig. (1)

Ubiquitylation of Sec23a. Huh7 cells were co-transfected with the pHA-Ubiquitin expression vector and the 3xFLAG-Sec23a vector for 48 h and then subjected to cycloheximide treatment for the indicated time. Cell lysates were immunoprecipitated by a mouse monoclonal anti-HA antibody to selectively pull down ubiquitinated proteins and then the 3xFLAG-Sec23a was revealed by a mouse monoclonal anti-FLAG antibody. Total lysates were checked by Western Blot with the indicated antibodies as control of the input samples.

Fig. (2)

Ubiquitylation of Sec23a in Hek293T cells. (A) SDS-PAGE and Coomassie-staining of Huh7 (H) and Hek293T (K) cells transiently transfected with 3X-FLAG-Sec23a at 48 h of expression. Total extracts (Lysates), the FLAG-immunoprecipitated proteins and the unbound proteins are showed as steps of the 3xFLAG-Sec23a purification. (B) Hek293T cells were co-transfected with the pHA-Ubiquitin expression vector and the 3xFLAG-Sec23a vector. Cell lysates were immunoprecipitated and checked by Western Blot with the indicated antibodies as in (Fig. 1).

Fig. (3)

Sec23a is ubiquitylated on cys432 and cys449. (A) Schematic representation of the Gly-Gly remnant left on ubiquitinated residues after trypsin digestion of the C-terminus of the linked ubiquitin (B;C) Output view of the software used for the search of the Gly-Gly modified peptides of Sec23a. The sequence and the MS/MS fragmentation peaks of the peptides containing the Gly-Gly-Cys 432 (B) and Gly-Gly-Cys 449 (C) are shown. (D) Extracted ion chromatograms corresponding to the Cys-modified peptide species.

Fig. (4)

Domain structure and sequence homology of Sec23a proteins. (A) Schematic representation of the structural domains of Sec23a (B) Protein multiple alignment obtained by HomoloGene (https://www.ncbi.nlm.nih.gov/homologene) of Sec23a proteins derived from various species. Conserved Cys 432 and Cys 449 are highlighted in red.

Fig. (5)

Sec23a ubiquitylation affects COPII vesicles formation. Huh7 cells seeded on glass coverslips were transiently transfected with the wilde type Sec23a-GFP or the mutated isoforms (C432A-Sec23a-GFP or C449A-Sec23a-GFP) and after 48 h of transfection processed for immunofluorescence with Sec16 specific antibody. The mean number of Sec23a-GFP positive spots (mean ± SD) and the percent of Sec23a-GFP pixels colabeling with Sec16a pixels (mean ± SD) are reported in the Sec23a-GFP panels and in the merged panels respectively.