AUTHOR=Schoberer Jennifer , Izadi Shiva , Kierein Carolina , Vavra Ulrike , König-Beihammer Julia , Ruocco Valentina , Grünwald-Gruber Clemens , Castilho Alexandra , Strasser Richard TITLE=The tobacco GNTI stem region harbors a strong motif for homomeric protein complex formation JOURNAL=Frontiers in Plant Science VOLUME=14 YEAR=2023 URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2023.1320051 DOI=10.3389/fpls.2023.1320051 ISSN=1664-462X ABSTRACT=Introduction

The Golgi apparatus of plants is the central cellular organelle for glycan processing and polysaccharide biosynthesis. These essential processes are catalyzed by a large number of Golgi-resident glycosyltransferases and glycosidases whose organization within the Golgi is still poorly understood.

Methods

Here, we examined the role of the stem region of the cis/medial Golgi enzyme N-acetylglucosaminyltransferase I (GNTI) in homomeric complex formation in the Golgi of Nicotiana benthamiana using biochemical approaches and confocal microscopy.

Results

Transient expression of the N-terminal cytoplasmic, transmembrane, and stem (CTS) regions of GNTI leads to a block in N-glycan processing on a co-expressed recombinant glycoprotein. Overexpression of the CTS region from Golgi α-mannosidase I, which can form in planta complexes with GNTI, results in a similar block in N-glycan processing, while GNTI with altered subcellular localization or N-glycan processing enzymes located further downstream in the Golgi did not affect complex N-glycan processing. The GNTI-CTS-dependent alteration in N-glycan processing is caused by a specific nine-amino acid sequence motif in the stem that is required for efficient GNTI-GNTI interaction.

Discussion

Taken together, we have identified a conserved motif in the stem region of the key N-glycan processing enzyme GNTI. We propose that the identified sequence motif in the GNTI stem region acts as a dominant negative motif that can be used in transient glycoengineering approaches to produce recombinant glycoproteins with predominantly mannosidic N-glycans.