Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.
Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.
Faik
A
A
McGill University, Department of Biology, 1025 Av. Dr. Penfield, Montreal, QC, H3A 1B1, Canada. faik@pilot.msu.edu
Desveaux
D
D
MacLachlan
G
G
D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D017360:Arabidopsis / Q000276:immunology; D029681:Arabidopsis Proteins; D001692:Biological Transport / Q000187:drug effects; D019607:Brassicaceae / Q000737:chemistry* / Q000166:cytology; D002592:Cetomacrogol / Q000494:pharmacology; D011232:Chemical Precipitation; D003429:Cross Reactions; D004220:Disulfides / Q000378:metabolism; D005638:Fruit / Q000737:chemistry* / Q000166:cytology; D005690:Galactose / Q000378:metabolism; D005936:Glucans; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013379:Substrate Specificity; D014530:Uridine Diphosphate / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*; D014617:Vacuoles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014990:Xylans
D000906:Antibodies; D029681:Arabidopsis Proteins; D004220:Disulfides; D005936:Glucans; D006023:Glycoproteins; D010455:Peptides; D010940:Plant Proteins; D011134:Polysaccharides; C106959:RGP1 protein, Arabidopsis; D014539:Uridine Diphosphate Sugars; D014990:Xylans; D014531:Uridine Diphosphate Galactose; C029353:xyloglucan; D014530:Uridine Diphosphate; D002592:Cetomacrogol; D005690:Galactose