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Protein N- glycosylation is one of the major post-translational modifications in eukaryotic cells. In lower unicellular eukaryotes, the known functions of N- glycans are predominantly in protein folding and quality control within the lumen of the endoplasmic reticulum (ER). In multicellular organisms, complex N- glycans are important for developmental programs and immune responses. However, little is known about the functions of complex N- glycans in plants. Formed in the Golgi apparatus, plant complex N- glycans have structures distinct from their animal counterparts due to a set of glycosyltransferases unique to plants. Severe basal underglycosylation in the ER lumen induces misfolding of newly synthesized proteins, which elicits the unfolded protein response (UPR) and ER protein quality control (ERQC) pathways. The former promotes higher capacity of proper protein folding and the latter degradation of misfolded proteins to clear the ER. Although our knowledge on plant complex N- glycan functions is limited, genetic studies revealed the importance of complex N- glycans in cellulose biosynthesis and growth under stress. [ABSTRACT FROM AUTHOR]

Summary: The oligosaccharyltransferase (OT) complex catalyzes N‐glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity‐tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super‐expression platform. Mass‐spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission‐electron microscopy showed that STT3a becomes incorporated into OT–ribosome super‐complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a. [ABSTRACT FROM AUTHOR]

We analyzed plant-derived α1,4-fucosyltransferase ( FucTc) homologs by reporter fusions and focused on representatives of the Brassicaceae and Solanaceae. Arabidopsis thaliana AtFucTc-green fluorescent protein ( GFP) or tomato LeFucTc-GFP restored Lewis-a formation in a fuctc mutant, confirming functionality in the trans-Golgi. AtFucTc-GFP partly accumulated at the nuclear envelope ( NE) not observed for other homologs or truncated AtFucTc lacking the N-terminus or catalytic domain. Analysis of At/ LeFucTc-GFP swap constructs with exchanged cytosolic, transmembrane and stalk ( CTS), or only the CT regions, revealed that sorting information resides in the membrane anchor. Other domains of AtFuctc also contribute, since amino-acid changes in the CT region strongly reduced but did not abolish NE localization. By contrast, two N-terminal GFP copies did, indicating localization at the inner nuclear membrane ( INM). Tunicamycin treatment of AtFucTc-GFP abolished NE localization and enhanced overlap with an endosomal marker, suggesting involvement of N-glycosylation. Yet neither expression in protoplasts of Arabidopsis N-glycosylation mutants nor elimination of the N-glycosylation site in AtFucTc prevented perinuclear accumulation. Disruption of endoplasmic reticulum ( ER)-to-Golgi transport by co-expression of Sar1( H74L) trapped tunicamycin-released AtFucTc-GFP in the ER, however, without NE localization. Since recovery after tunicamycin-washout required de novo-protein synthesis, our analyses suggest that AtFucTc localizes to the NE/ INM due to interaction with an unknown (glyco)protein. [ABSTRACT FROM AUTHOR]

We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision. [ABSTRACT FROM AUTHOR]