TAILIEUCHUNG - Báo cáo khoa học: Insertion of the plant photosystem I subunit G into the thylakoid membrane In vitro and in vivo studies of wild-type and tagged versions of the protein

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane a-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosys-tem I subunit G (PSI-G). | iFEBS Journal Insertion of the plant photosystem I subunit G into the thylakoid membrane In vitro and in vivo studies of wild-type and tagged versions of the protein Lisa Rosgaard Agnieszka Zygadlo Henrik Vibe Scheller Alexandra Mantt and Poul Erik Jensen Plant Biochemistry Laboratory Department of Plant Biology The RoyalVeterinary AgriculturalUniversity Frederiksberg Denmark Keywords His-tag membrane topology photosystem I Strep-tag transgenic Arabidopsis Correspondence P. E. Jensen Plant Biochemistry Laboratory Department of Plant Biology The Royal Veterinary AgriculturalUniversity 40 Thorvaldsensvej DK-1871 Frederiksberg C Denmark Fax 45 35283333 Tel 45 35283354 E-mail peje@ Present address Novozymes A S Starch R D Laurentsvej 55 Bagsvmrd Denmark tPresent address Molecular Immunology Group Cancer Sciences Division Southampton General Hospital Mailpoint 824 Tremona Road Southampton SO16 6YD UK Note L. Rosgaard and A. Zygadlo contributed equally to this work Received 9 May 2005 revised 14 June 2005 accepted 17 June 2005 doi Subunit G of photosystem I is a nuclear-encoded protein predicted to form two transmembrane a-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G PSI-G . However the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G .

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