Vesicle transport and processing of the precursor to 2S albumin in pumpkin.
Cell fractionation of pulse-chase-labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles. The primary structure of the precursor (16,578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N-terminus indicates that the precursor is a pre-proprotein that is converted into pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off cotranslationally on the C-terminal side of alanine residue 22 of prepro2S albumin. By contrast, posttranslational cleavages occur on the C-terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.
Hara-Hishimura
I
I
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Takeuchi
Y
Y
Inoue
K
K
Nishimura
M
M
D000418:Albumins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005638:Fruit / Q000378:metabolism / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D012639:Seeds / Q000378:metabolism* / Q000648:ultrastructure; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000378:metabolism / Q000648:ultrastructure
D000418:Albumins; D011498:Protein Precursors; D021382:Protein Sorting Signals