Isolation of a putative receptor for KDEL-tailed cysteine proteinase (SH-EP) from cotyledons of Vigna mungo seedlings.
SH-EP is the major papain-type proteinase expressed in cotyledons of germinated Vigna mungo seeds. The proteinase possesses a KDEL sequence at the C-terminus although the mature form of SH-EP is localized in vacuoles. It has also been shown that the proform of SH-EP is accumulated at the edge or middle region of the endoplasmic reticulum, and the accumulated proSH-EP is directly transported to vacuoles via the KDEL-tailed cysteine proteinase-accumulating vesicle, KV. In this study, to address the transport machinery of proSH-EP through KV, putative receptor for proSH-EP was isolated from membrane proteins of cotyledons of V. mungo seedlings using a proSH-EP-immobilized column. The deduced amino acid sequence from cDNA to the protein revealed that the putative receptor for proSH-EP is a member of vacuolar sorting receptor, VSR, that is known to be localized in the Golgi-complex and/or clathrin coated vesicle. We carried out subcellular fractionation of cotyledon cells and subsequently conducted SDS-PAGE/immunoblotting and immunocytochemistry with anti-V. mungo VSR (VmVSR) or SH-EP antibody. The results showed that VmVSR is co-localized in the fraction of the gradient in which KV existed.
Tsuru-Furuno
A
A
Department of Biological Sciences, Tokyo Metropolitan University, Minami-osawa 1-1, Hachioji, Tokyo, 192-0397 Japan.
Okamoto
T
T
Minamikawa
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D007887:Fabaceae / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; C449139:KDEL-tailed cysteine proteinase receptor, Vigna mungo; D010940:Plant Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface