The role of early-Golgi N-glycosylation enzyme complexes

The Golgi apparatus is the central biosynthetic organelle of the secretory pathway and as such plays a pivotal role in the transport and concomitant modification of proteins and polysaccharides in all eukaryotes. The step-wise modification of protein-bound asparagine-linked oligosaccharides (N-glycans) in the stacks of the plant Golgi apparatus is the outcome of multiple sequentially acting glycosidases and glycosyltransferases, which are asymmetrically distributed across the cis-, medial- and trans-Golgi cisternae. It is commonly believed that the highly-defined cisternal organisation of Golgi-resident enzymes is necessary and essential for the functionality of the Golgi. How this asymmetric Golgi distribution is established and maintained is currently unknown and the mechanisms that regulate Golgi enzyme organisation and interaction with cargo molecules are not understood. By using the non-invasive biophysical FRET-FLIM method we recently discovered that several cis/medial-Golgi enzymes involved in N-glycan processing assemble into homo- and heterodimeric complexes in planta. We hypothesise that the assembly of discrete subsets of enzymes into functionally relevant enzyme complexes via protein-protein interactions could serve to maintain the compartment-specific Golgi (protein) organisation and/or may be a functional tool to regulate N-glycan processing. So far the biological significance of enzyme complex formation in the plant Golgi is speculative and it is also not clear whether the existing homo- and heterodimers are part of a larger oligomeric enzyme complex. This project aims to characterise an N-glycan processing enzyme complex that resides in the cis/medial-cisternae of plant Golgi stacks. We will utilise complementary cell biological, genetic, biochemical and proteomics approaches to identify its biological role in the development and physiology of plants and unravel its functional relevance in the maintenance of normal Golgi functions such as the specific protein glycosylation events in plant cells. To investigate the potential formation of a multi-enzyme complex in planta we will develop and apply 3- colour FRET combined multiphoton-induced FLIM to visualise multiple interactions at the same time in the same organelle. Together, these experiments will elucidate the composition and the biological significance of the identified enzyme complex and provide novel insights into the organisation of the plant Golgi apparatus and its resident proteins at a structural and functional level.

The plant Golgi apparatus is an important cellular organelle consisting of individual stacks of membrane-enclosed disks. These disks are filled with enzymes that modify sugar chains of e.g. proteins ("glycosylation"). The aim of this project was to characterise a Golgi-localised protein complex in plants, which consists of three different glycosylation enzymes. Using cell biological, genetic and biochemical methods, the biological role or functional importance of this enzyme complex for the maintenance of normal Golgi functions, such as the modification of sugar chains, and the development of the plant were investigated. For this purpose, the model plants Nicotiana sp. and Arabidopsis thaliana were studied. One of the first findings was that a single amino acid in the membrane domain of an important enzyme called GnTI, a glycosyltransferase that controls the production of complex sugar chains, is responsible for its positioning within the Golgi stack. Its luminal domain, however, is responsible for the interaction with itself and other glycosylation enzymes. We also developed a non-invasive microscopic method called "2-color FRET-FLIM" to simultaneously visualise several protein interactions in the same organelle in living cells. In the course of the project, this method has successfully been implemented in combination with human cells and is currently being adapted for the use of plant cells in order to fully elucidate the structure of the above- mentioned Golgi-localised enzyme complex. The characterisation of the interactome of GnTI also revealed an interaction with an ER-alpha-mannosidase called MNS3, which is retained in the Golgi apparatus due to a specific amino acid signal. This signal is not similar to any of the hitherto known localisation signals in plants, mammals and yeasts. It is responsible for the fact that in the endoplasmic reticulum (ER) the degradation of inactive glycoproteins, which endanger the survival of the cell, is not disturbed by the presence and activity of MNS3 in the ER.