Accumulation and proteolytic processing of vicilin deletion-mutant proteins in the leaf and seed of transgenic tobacco.
Vicilin, a 7S globulin of Pisum sativum L. seed, accumulates in protein-storage vacuoles (protein bodies) of cotyledonary storage-parenchyma cells. The synthesis and proteolytic processing of various genetically engineered proteins within the leaf and seed of a heterologous (tobacco, Nicotiana tabacum L.) host was examined. A modified vicilin gene, in which the DNA sequence corresponding to the signal peptide was removed, resulted in a polypeptide of 50 kDa in the tobacco leaf and seed; none of the normal proteolytic cleavage products characteristic of expression of an unmodified vicilin gene were obtained. Likewise, no vacuolar accumulation of this mutant vicilin occurred in leaf protoplasts, which is also supportive of the predicted cytosolic localization for this protein. In-frame deletions were made within the region of the vicilin gene encoding the mature protein, to eliminate the N-terminal 28 and 121 amino acids and the C-terminal 69 residues, while maintaining an intact signal peptide. All of these "mature" deletion-mutant proteins were accumulated to only low levels in the host, but exhibited the predicted molecular weight and underwent some normal proteolytic processing in the seed. Mutant vicilin proteins having deletions in either the N-terminus (delta NT 121) or C-terminus (delta CT 69) were not found in appreciable amounts within the vacuolar fraction of transgenic tobacco leaf protoplasts, perhaps due to protein degradation in this compartment. Compared with the intact vicilin, oligomer assembly of the C-terminal deletion-mutant protein was disrupted in leaf cells, which may have further affected protein stability.(ABSTRACT TRUNCATED AT 250 WORDS)
Kermode
A R
AR
Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada.
Fisher
S A
SA
Polishchuk
E
E
Wandelt
C
C
Spencer
D
D
Higgins
T J
TJ
D001483:Base Sequence; D015153:Blotting, Western; D017353:Gene Deletion; D017343:Genes, Plant; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011499:Protein Processing, Post-Translational; D011523:Protoplasts / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055314:Seed Storage Proteins; D012639:Seeds / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C015575:vicilin protein, plant