Dissecting the ER-Golgi interface using Arabidopsis MNS3

The project Dissecting the ER-Golgi interface using Arabidopsis MNS3 centres on the study of the Endoplasmic Reticulum (ER) alpha-mannosidase MNS3 from the model plant Arabidopsis thaliana. This enzyme is part of the asparagine (N)-linked protein glycosylation machinery, which is a (biologically) important protein modification in eukaryotes and mainly takes place in the ER and Golgi apparatus. MNS3 generates an oligo-mannosidic N-glycan structure that is mainly found on ER- resident glycoproteins, but surprisingly the enzyme itself does not reside in the ER. Our preliminary data show that MNS3 concentrates in a compartment of the Golgi apparatus that is adjacent to the ER and appears resistant to common chemical and genetic means that cause the rapid breakdown of Golgi structures. Such unusual behaviour has only been observed for a few other Golgi proteins that potentially reside in the same Golgi compartment. We hypothesise that MNS3 together with these proteins resides in a so far uncharacterised membrane compartment within the ER-Golgi interface that may be functionally related to the ER-Golgi intermediate compartment in mammalian cells. This subcellular compartment potentially facilitates protein sorting and serves as a scaffold for Golgi biogenesis in plants. This project aims to (1) pinpoint the subcellular whereabouts of MNS3 by dissecting the so far ill-defined ER-Golgi interface in plants, (2) reconcile this peculiar subcellular localisation with its biosynthetic and biological function (typically performed in the ER), and (3) determine the underlying localisation mechanisms. We will use the model plants Nicotiana benthamiana and A. thaliana and apply advanced cell biological, biochemical and genetic tools to shed light on the functional relevance of this peculiar subcellular compartmentation of MNS3 in plants. We anticipate that the outcome of this project will provide cell biologists from all fields with a better understanding of the mechanisms that control the transport of proteins through the secretory pathway. Knowledge of these mechanisms and the regulation of associated biosynthetic functions in plants is insofar important as plants bear a huge biotechnological and industrial potential for the production of pharmaceutically-relevant proteins (i.e. antibodies) that very often are highly N- glycosylated and transported through the secretory pathway.

The protein MNS3 from the model plant Arabidopsis thaliana is a mannosidase that plays a key role in the processing of N-glycans. N-glycans are carbohydrate (sugar) chains that are attached to proteins during glycosylation. Glycosylation is one of the most widespread forms of protein modification in all eukaryotes. It influences many biological processes and takes place mainly in the endoplasmic reticulum (ER) and the Golgi apparatus, two specialised membrane compartments within the cell. MNS3 produces a sugar structure that is mainly found on glycoproteins in the ER, but surprisingly the enzyme is not localised in the ER. The aim of this project was to determine the exact localisation of MNS3 in plant cells, particularly in the region between the ER and the Golgi. We also wanted to understand how this localisation is compatible with the biosynthetic functions of MNS3 and how MNS3 reaches its steady-state location in the cell. We discovered a previously unknown signal that is responsible for the localisation and retention of MNS3 in the Golgi. This Golgi localisation results in strict spatial separation from the ER-localised mannose processing steps that generate the glycan signal for the labelling and subsequent degradation of terminally misfolded glycoproteins. This prevents random (as opposed to targeted) degradation of glycoproteins in the ER. We also used a variety of methods to identify proteins that can bind to this new signalling motif. We obtained a number of candidates that are localised in the ER and Golgi and have known regulatory functions in the selection and transport of cargo proteins. Among these candidates, we identified an important regulatory protein with a function in protein transport between ER and Golgi. Overexpression of this protein led to an increase in the levels of several co-expressed proteins. Arabidopsis mutants lacking this protein showed an altered morphology of the Golgi apparatus and an up-regulated expression of a gene involved in the formation of transport vehicles between the ER and Golgi. In conclusion, our data show that an increase in the identified protein triggers a secretory response in plant cells, suggesting a novel regulatory function of this protein. These findings allow the further development of molecular tools for the targeted enhancement of protein expression in plants, which have enormous biotechnological and industrial potential for the production of recombinant, pharmaceutically relevant glycoproteins.