RESUMEN

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.

Asunto(s)

Fabaceae/metabolismo , Concentración de Iones de Hidrógeno , Proteínas de Plantas/aislamiento & purificación , Receptores de Superficie Celular/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , ARN Mensajero/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo