A blue native-PAGE analysis of membrane protein complexes in Clostridium thermocellum.
BACKGROUND
Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology.


RESULTS
A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation.


CONCLUSIONS
Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
Yanfeng
Y
Peng
Yuanming
Y
Luo
Tingting
T
Yu
Xinping
X
Xu
Keqiang
K
Fan
Youbao
Y
Zhao
Keqian
K
Yang
D001426:Bacterial Proteins / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001426:Bacterial Proteins; D008565:Membrane Proteins
Trends Cell Biol. 2007 Dec;17(12):600-8
17983748
Biochemistry. 2008 Sep 30;47(39):10354-64
18771296
Structure. 2002 Oct;10(10):1415-23
12377127
BMC Syst Biol. 2010 Mar 22;4:31
20307315
Trends Biochem Sci. 2002 Mar;27(3):154-60
11893513
Proc Natl Acad Sci U S A. 2007 Oct 2;104(40):15888-93
17893339
Nucleic Acids Res. 2001 Jan 1;29(1):22-8
11125040
J Mol Biol. 2009 May 29;389(1):17-33
19327365
Science. 2007 Sep 7;317(5843):1387-90
17673622
Biochemistry. 2010 Mar 23;49(11):2409-15
20148520
Nat Protoc. 2006;1(1):418-28
17406264
Appl Microbiol Biotechnol. 2007 Feb;74(2):422-32
17124583
Eur J Biochem. 2003 Feb;270(4):736-44
12581213
J Biol Chem. 2004 Feb 6;279(6):5000-7
14610094
J Biol Chem. 1998 Dec 18;273(51):34247-54
9852088
Anal Biochem. 2006 Jul 1;354(1):70-7
16707089
Structure. 2000 Nov 15;8(11):1215-26
11080643
Infect Immun. 2002 Mar;70(3):1254-9
11854208
J Bacteriol. 2004 Mar;186(6):1758-68
14996807
Biochemistry. 1979 Apr 17;18(8):1413-22
218620
J Biol Chem. 2002 Aug 23;277(34):31228-36
12048195
Arch Biochem Biophys. 1983 Sep;225(2):576-85
6137995
Microbiol Mol Biol Rev. 2008 Jun;72(2):317-64, table of contents
18535149
Res Microbiol. 2009 Nov;160(9):652-9
19744556
Anal Biochem. 1976 May 7;72:248-54
942051
Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2801-6
9501170
Proteins. 2000 Jun 1;39(4):271-7
10813810
J Biol Inorg Chem. 2007 Jun;12(5):615-30
17541801
Anal Biochem. 1991 Dec;199(2):223-31
1812789
Electrophoresis. 2006 Jul;27(14):2984-95
16718645
Plant Physiol. 2004 Jan;134(1):470-81
14730074
Proc Int Conf Intell Syst Mol Biol. 1998;6:175-82
9783223
EMBO J. 2000 Sep 1;19(17):4473-84
10970841
J Mol Biol. 2009 Feb 13;386(1):37-59
18801369
J Biol Chem. 2000 Jun 16;275(24):18093-8
10764779
J Proteome Res. 2010 Jul 2;9(7):3730-40
20521846
J Biol Chem. 2001 Jun 8;276(23):20136-43
11274185
Biochimie. 2007 Jan;89(1):145-57
17084501
J Biol Chem. 1993 Jun 5;268(16):11610-6
8505293
J Bioenerg Biomembr. 2005 Dec;37(6):405-10
16691473
Biosci Biotechnol Biochem. 2010;74(7):1391-5
20622460
Nat Struct Mol Biol. 2005 Jul;12(7):575-81
15951817
Crit Rev Microbiol. 1994;20(1):13-56
8185833
Nature. 2007 Mar 8;446(7132):213-6
17322901
J Bacteriol. 1997 Feb;179(4):1274-9
9023212
Mol Cell Proteomics. 2007 Mar;6(3):460-78
17164403
J Mol Biol. 2009 Oct 23;393(2):425-34
19666033
J Biol Chem. 2005 Oct 14;280(41):34409-19
16079137
J Bacteriol. 2009 Jan;191(1):203-9
18952792
Nat Struct Mol Biol. 2010 Mar;17(3):373-8
20173764
J Bacteriol. 2007 Oct;189(19):7154-8
17644609
J Mol Biol. 2001 Jan 19;305(3):567-80
11152613
Proteomics. 2005 Feb;5(3):746-57
15682463
Microbiol Mol Biol Rev. 2005 Mar;69(1):124-54
15755956
Nucleic Acids Res. 2000 Jan 1;28(1):33-6
10592175
Biosci Biotechnol Biochem. 2001 Dec;65(12):2763-72
11826975
J Mol Biol. 1998 Oct 30;283(3):695-704
9784377
Biochim Biophys Acta. 2001 May 1;1505(1):75-81
11248190
Mol Microbiol. 2008 Nov;70(4):780-2
18990179
J Proteome Res. 2006 Dec;5(12):3268-76
17137328
J Biol Chem. 2003 Aug 15;278(33):30806-12
12794081
BMC Microbiol. 2009 Aug 10;9:162
19664283
Cell. 1998 Feb 6;92(3):291-4
9476889
Science. 2005 Apr 29;308(5722):654-9
15802565
J Struct Biol. 2004 Apr-May;146(1-2):123-9
15037243
Prog Lipid Res. 2002 Sep;41(5):407-35
12121720
EMBO J. 2003 May 1;22(9):1959-68
12727864