Dissecting the ER-Golgi interface using Arabidopsis MNS3

N-linked protein glycosylation is one of the most prominent types of protein modification in eukaryotic cells,which plays an important role in diverse biological processes. Protein-linked N-glycans are modifiedthrough the sequential action of processing glycosidases and glycosyltransferases that strictly segregateinto distinct compartments along the ER-Golgi secretory route according to their function in the N-glycanprocessing pathway. The Arabidopsis ER-alpha-mannosidase I (MNS3) generates an oligomannosidic N-glycan structure that is characteristically found on ER-resident glycoproteins. However, the enzyme exclusively resides in the Golgi apparatus at steady-state. Notably, MNS3 remains on dispersed punctate structures when subjected to different approaches that commonly result in the relocation of Golgi enzymes to the ER. Responsible for this rare behavior is an amino acid signal motif (LPYS) within the cytoplasmic tail of MNS3 that acts as a specific Golgi retention signal. To identify proteins that might bind to this novel signal motif, we used different proteomics approaches in Arabidopsis thaliana and Nicotiana benthamiana, which yielded a set of ER- and Golgi-localised candidate proteins with known regulatory functions in cargo selection and transport. Among these candidates, we identified a Golgi-resident Rab GTPase that is involved in ER-Golgi trafficking in plants. This Rab GTPase not only interacted with MNS3 in planta, but also increased MNS3 expression when both recombinant proteins were heterologously co-expressed in N. benthamiana. Overexpression of the Rab GTPase also increased the secretion of a non-plant secretory protein into the apoplastic fluid. Arabidopsis mutants lacking the identified Rab GTPase showed Golgi stacks with reduced cisternae length and width and transcriptional upregulation of SEC31A. We hypothesise that an increase in Rab GTPase levels triggers a secretory response in plant cells, which is currently under investigation.