A novel membrane protein that is transported to protein storage vacuoles via precursor-accumulating vesicles.
A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long alpha-helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73 Delta Psi) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 Delta Psi from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.
Mitsuhashi
N
N
Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Hayashi
Y
Y
Koumoto
Y
Y
Shimada
T
T
Fukasawa-Akada
T
T
Nishimura
M
M
Hara-Nishimura
I
I
D000595:Amino Acid Sequence; D028464:Cucurbita / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D014617:Vacuoles / Q000502:physiology*
C445098:CMMP73 protein, Cucurbita maxima; D008565:Membrane Proteins; D010940:Plant Proteins; D011498:Protein Precursors