O-glycosylation of a precursor to a sweet potato vacuolar protein, sporamin, expressed in tobacco cells.
Sporamin, a vacuolar protein of the sweet potato, is synthesized as a precursor that contains signal peptide and an N-terminal propeptide that functions as a vacuolar targeting determinant. Sporamin, when expressed in tobacco cells, migrated as smeared bands on an SDS-polyacrylamide gel. The smearing was due to O-glycosylation of the precursor to sporamin. The smeared bands were stained by a glycan-specific stain but no N-glycosylation site was found in the amino acid sequence of the precursor to sporamin. The glycan attached to sporamin contained galactose and arabinose as major sugar components. Mutations that altered the Pro36 or Ser39 residue of the precursor to sporamin prevented glycosylation of the protein, and analysis by semiquantitative Edman degradation suggested that a glycan moiety was attached to Pro36 and, possibly, to Ser39. Pulse-labeling and cell-fractionation experiments revealed that the O-glycosylation of the precursor to sporamin occurred in the Golgi apparatus. Thus, this modification serves as a good marker of the transport from the endoplasmic reticulum (ER) to the Golgi apparatus of the precursor to sporamin. Treatment of transformed tobacco cells with brefeldin A (BFA) caused the intracellular accumulation of prosporamin that did not migrate as smeared bands. Thus, it appeared that BFA inhibited the transport of the precursor to sporamin to the Golgi apparatus. This result provides the first biochemical evidence that BFA inhibits transport from the ER to the Golgi apparatus in plant cells.
Matsuoka
K
K
Laboratory of Biochemistry, School of Agricultural Sciences, Nagoya University, Japan.
Watanabe
N
N
Nakamura
K
K
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000595:Amino Acid Sequence; D001483:Base Sequence; D020126:Brefeldin A; D000001:Calcimycin / Q000494:pharmacology; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D017931:DNA Primers; D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000096:biosynthesis*; D010947:Plants, Toxic; D017354:Point Mutation; D011392:Proline; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012694:Serine; D014026:Nicotiana / Q000378:metabolism; D014162:Transfection; D014617:Vacuoles / Q000378:metabolism*; D014675:Vegetables / Q000378:metabolism*
D002241:Carbohydrates; D003517:Cyclopentanes; D017931:DNA Primers; D006023:Glycoproteins; D010446:Peptide Fragments; D010940:Plant Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C067142:sporamin protein, Ipomoea batatas; D020126:Brefeldin A; D000001:Calcimycin; D012694:Serine; D015082:2,2'-Dipyridyl; D008985:Monensin; D011392:Proline