Identification of the pore-forming region of the outer chloroplast envelope protein OEP16.
The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.
Steinkamp
T
T
Fachbereich Biologie/Chemie, Universität Osnabrück, D-49034 Osnabrück, Germany.
Hill
K
K
Hinnah
S C
SC
Wagner
R
R
Röhl
T
T
Pohlmeyer
K
K
Soll
J
J
D000588:Amines / Q000378:metabolism; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry*; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D004926:Escherichia coli; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism
D000588:Amines; D000596:Amino Acids; D060365:Chloroplast Proteins; D007473:Ion Channels; D010940:Plant Proteins; D011994:Recombinant Proteins; D012694:Serine; D003300:Copper; D003545:Cysteine; C029892:cupric chloride