Multiple vacuolar sorting determinants exist in soybean 11S globulin.
The sorting determinants of glycinin, a soybean (Glycine max) 11S globulin, which mediates protein targeting to the protein storage vacuole (PSV), were investigated in maturing soybean cotyledons by transient expression assays. A C-terminal stretch of 10 amino acids of A1aB1b, a glycinin group I subunit, was sufficient to direct green fluorescent protein (GFP) to the PSV. This peptide may correspond to a C-terminal vacuolar sorting determinant (ctVSD). Because functional inhibition of this putative ctVSD of A1aB1b did not block PSV sorting of A1aB1b, we used the three-dimensional structure of A1aB1b to identify candidates for a sequence-specific determinant (ssVSD). We found that the sequence downstream of disordered region 4 could direct GFP to the PSV and that Ile-297 is critical for sorting. However, functional inhibition of the ctVSD, combined with the Ile297Gly mutation, did not abolish the vacuolar sorting of A1aB1b, suggesting that A1aB1b has a third sorting determinant in addition to ctVSD and ssVSD. A glycinin group II subunit, A3B4, lacked a ctVSD but contained a VSD reminiscent of an ssVSD and an additional sorting determinant. We also demonstrate, by expression of dominant negative mutants of small GTPases and drug treatment experiments, that the trafficking of A1aB1b is COPII vesicle-dependent and wortmannin- and brefeldin A-sensitive.
Maruyama
Nobuyuki
N
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Mun
Leong Ching
LC
Tatsuhara
Miyuki
M
Sawada
Machiko
M
Ishimoto
Masao
M
Utsumi
Shigeru
S
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D022181:COP-Coated Vesicles / Q000502:physiology; D018548:Cotyledon / Q000378:metabolism; D005916:Globulins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D021382:Protein Sorting Signals; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D015398:Signal Transduction; D030262:Soybean Proteins; D013025:Glycine max / Q000378:metabolism* / Q000648:ultrastructure; D014617:Vacuoles / Q000378:metabolism* / Q000648:ultrastructure; D000077191:Wortmannin
D000730:Androstadienes; D005916:Globulins; D010940:Plant Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D030262:Soybean Proteins; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; C011474:glycinin; D020823:ADP-Ribosylation Factor 1; D000077191:Wortmannin