In eukaryotes, class I a-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ERtype a-mannosidase I (MNS3) and the two Golgi-a-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected a1,2-mannose residue from Man 9 GlcNAc 2 , MNS1/2 readily removed three a1,2-mannose residues from Man 8 GlcNAc 2 . Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man 8 GlcNAc 2 accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man 9 GlcNAc 2 , demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I a-mannosidases in wildtype seedlings resulted in a similar root phenotype. These findings show that class I a-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuca1-4(Galb1-3) GlcNAc-R] structures. This process involves the sequential attachment of b1,3-galactose and a1,4-fucose residues by b1,3-galactosyltransferase and a1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, b1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana b1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring b1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with a1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of b1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.

Plant N-glycan processing enzymes are arranged along the early secretory pathway, forming an assembly line to facilitate the step-by-step modification of oligosaccharides on glycoproteins. Thus, these enzymes provide excellent tools to study signals and mechanisms, promoting their localization and retention in the endoplasmic reticulum (ER) and Golgi apparatus. Herein, we focused on a detailed investigation of amino acid sequence motifs present in their short cytoplasmic tails in respect to ER export. Using site-directed mutagenesis, we determined that single arginine/lysine residues within the cytoplasmic tail are sufficient to promote rapid Golgi targeting of Golgiresident N-acetylglucosaminyltransferase I (GnTI) and a-mannosidase II (GMII). Furthermore, we reveal that an intact ER export motif is essential for proper in vivo function of GnTI. Coexpression studies with Sar1p provided evidence for COPII-dependent transport of GnTI to the Golgi. Our data provide evidence that efficient ER export of Golgi-resident plant N-glycan processing enzymes occurs through a selective mechanism based on recognition of single basic amino acids present in their cytoplasmic tails.

Summary N-glycosylation is one of the major post-translational modifications of proteins in eukaryotes; however, the processing reactions of oligomannosidic N-glycan precursors leading to hybrid-type and finally complex-type N-glycans are not fully understood in plants. To investigate the role of Golgi a-mannosidase II (GMII) in the formation of complex N-glycans in plants, we identified a putative GMII from Arabidopsis thaliana (AtGMII; EC 3.2.1.114) and characterized the enzyme at a molecular level. The putative AtGMII cDNA was cloned, and its deduced amino acid sequence revealed a typical type II membrane protein of 1173 amino acids. A soluble recombinant form of the enzyme produced in insect cells was capable of processing different physiologically relevant hybrid N-glycans. Furthermore, a detailed N-glycan analysis of two AtGMII knockout mutants revealed the predominant presence of unprocessed hybrid N-glycans. These results provide evidence that AtGMII plays a central role in the formation of complex N-glycans in plants. Furthermore, conclusive evidence was obtained that alternative routes in the conversion of hybrid N-glycans to complex N-glycans exist in plants. Transient expression of N-terminal AtGMII fragments fused to a GFP reporter molecule demonstrated that the transmembrane domain and 10 amino acids from the cytoplasmic tail are sufficient to retain a reporter molecule in the Golgi apparatus and that lumenal sequences are not involved in the retention mechanism. A GFP fusion construct containing only the transmembrane domain was predominantly retained in the ER, a result that indicates the presence of a motif promoting ER export within the last 10 amino acids of the cytoplasmic tail of AtGMII.

To ensure that aberrantly folded proteins are cleared from the endoplasmic reticulum (ER), all eukaryotic cells possess a mechanism known as endoplasmic reticulum-associated degradation (ERAD). Many secretory proteins are N-glycosylated, and despite some recent progress, little is known about the mechanism that selects misfolded glycoproteins for degradation in plants. Here, we investigated the role of Arabidopsis thaliana class I a-mannosidases (MNS1 to MNS5) in glycan-dependent ERAD. Our genetic and biochemical data show that the two ER-resident proteins MNS4 and MNS5 are involved in the degradation of misfolded variants of the heavily glycosylated brassinosteroid receptor, BRASSINOSTEROID INSENSITIVE1, while MNS1 to MNS3 appear dispensable for this ERAD process. By contrast, N-glycan analysis of different mns mutant combinations revealed that MNS4 and MNS5 are not involved in regular N-glycan processing of properly folded secretory glycoproteins. Overexpression of MNS4 or MNS5 together with ER-retained glycoproteins indicates further that both enzymes can convert Glc 0-1 Man 8-9 GlcNAc 2 into N-glycans with a terminal a1,6-linked Man residue in the C-branch. Thus, MNS4 and MNS5 function in the formation of unique N-glycan structures that are specifically recognized by other components of the ERAD machinery, which ultimately results in the disposal of misfolded glycoproteins.