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KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway
Author(s) -
Wang Pengwei,
Hummel Eric,
Osterrieder Anne,
Meyer Andreas J.,
Frigerio Lorenzo,
Sparkes Imogen,
Hawes Chris
Publication year - 2011
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/j.1365-313x.2011.04522.x
Subject(s) - endoplasmic reticulum , golgi apparatus , microbiology and biotechnology , transmembrane domain , transmembrane protein , biology , membrane protein , secretory pathway , green fluorescent protein , membrane topology , chemistry , biochemistry , membrane , gene , receptor
Summary We have identified two endoplasmic reticulum (ER)‐associated Arabidopsis proteins, KMS1 and KMS2, which are conserved among most species. Fluorescent protein fusions of KMS1 localised to the ER in plant cells, and over‐expression induced the formation of a membrane structure, identified as ER whorls by electron microscopy. Hydrophobicity analysis suggested that KMS1 and KMS2 are integral membrane proteins bearing six transmembrane domains. Membrane protein topology was assessed by a redox‐based topology assay (ReTA) with redox‐sensitive GFP and confirmed by a protease protection assay. A major loop domain between transmembrane domains 2 and 3, plus the N‐ and C‐termini were found on the cytosolic side of the ER. A C‐terminal di(tri)‐lysine motif is involved in retrieval of KMS1 and deletion led to a reduction of the GFP–KMS1 signal in the ER. Over‐expression of KMS1/KMS2 truncations perturbed ER and Golgi morphology and similar effects were also seen when KMS1/KMS2 were knocked‐down by RNA interference. Microscopy and biochemical experiments suggested that expression of KMS1/KMS2 truncations inhibited ER to Golgi protein transport.