title: KMS1 and KMS2, two plant endoplasmic reticulum proteins involved in the early secretory pathway.
abstract: 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.
authors.affiliation: School of Life Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, UK.
authors.forename: Pengwei
authors.initials: P
authors.lastname: Wang
authors.forename: Eric
authors.initials: E
authors.lastname: Hummel
authors.forename: Anne
authors.initials: A
authors.lastname: Osterrieder
authors.forename: Andreas J
authors.initials: AJ
authors.lastname: Meyer
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Frigerio
authors.forename: Imogen
authors.initials: I
authors.lastname: Sparkes
authors.forename: Chris
authors.initials: C
authors.lastname: Hawes
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure*; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D021381:Protein Transport; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D055571:Secretory Pathway; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D049452:Green Fluorescent Proteins