An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in A rabidopsis culture cells
Summary Vacuolar sorting receptors ( VSR s) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late G olgi or trans ‐ G olgi network ( TGN ) for vacuolar transport via the pre‐vacuolar compartment ( PVC ). However, little is known about VS...
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Published in | The Plant journal : for cell and molecular biology Vol. 75; no. 6; pp. 1003 - 1017 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
01.09.2013
|
Online Access | Get full text |
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Summary: | Summary
Vacuolar sorting receptors (
VSR
s) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late
G
olgi or
trans
‐
G
olgi network (
TGN
) for vacuolar transport via the pre‐vacuolar compartment (
PVC
). However, little is known about
VSR
cargo proteins in plants. Here we developed and tested an
in vivo
expression system for the identification of
VSR
cargos which is based on the premise that the expressed
N
‐terminus of
VSR
s will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic
A
rabidopsis culture cell lines expressing VSR N‐terminal binding domains (VSRNTs) were shown to secrete truncated
VSR
s (
BP
80
NT
, At
VSR
1
NT
and At
VSR
4
NT
) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with
VSR
s. The screening strategy described here should be applicable to all
VSR
s and will help identify and study cargo proteins for individual
VSR
proteins. This method should be useful for both cargo identification and protein–protein interaction
in vivo
. |
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ISSN: | 0960-7412 1365-313X |
DOI: | 10.1111/tpj.12257 |