Novel multiprotein complexes identified in the hyperthermophilic archaeon Pyrococcus furiosus by non-denaturing fractionation of the native proteome.
Virtually all cellular processes are carried out by dynamic molecular assemblies or multiprotein complexes, the compositions of which are largely undefined. They cannot be predicted solely from bioinformatics analyses nor are there well defined techniques currently available to unequivocally identify protein complexes (PCs). To address this issue, we attempted to directly determine the identity of PCs from native microbial biomass using Pyrococcus furiosus, a hyperthermophilic archaeon that grows optimally at 100 degrees C, as the model organism. Novel PCs were identified by large scale fractionation of the native proteome using non-denaturing, sequential column chromatography under anaerobic, reducing conditions. A total of 967 distinct P. furiosus proteins were identified by mass spectrometry (nano LC-ESI-MS/MS), representing approximately 80% of the cytoplasmic proteins. Based on the co-fractionation of proteins that are encoded by adjacent genes on the chromosome, 106 potential heteromeric PCs containing 243 proteins were identified, only 20 of which were known or expected. In addition to those of unknown function, novel and uncharacterized PCs were identified that are proposed to be involved in the metabolism of amino acids (10), carbohydrates (four), lipids (two), vitamins and metals (three), and DNA and RNA (nine). A further 30 potential PCs were classified as tentative, and the remaining potential PCs (13) were classified as weakly interacting. Some major advantages of native biomass fractionation for PC identification are that it provides a road map for the (partial) purification of native forms of novel and uncharacterized PCs, and the results can be utilized for the recombinant production of low abundance PCs to provide enough material for detailed structural and biochemical analyses.
Menon
Angeli Lal
AL
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Poole
Farris L
FL
Cvetkovic
Aleksandar
A
Trauger
Sunia A
SA
Kalisiak
Ewa
E
Scott
Joseph W
JW
Shanmukh
Saratchandra
S
Praissman
Jeremy
J
Jenney
Francis E
FE
Wikoff
William R
WR
Apon
John V
JV
Siuzdak
Gary
G
Adams
Michael W W
MW
D000596:Amino Acids / Q000378:metabolism; D019843:Archaeal Proteins / Q000032:analysis* / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods*; D003593:Cytoplasm / Q000378:metabolism; D046912:Multiprotein Complexes / Q000032:analysis*; D011489:Protein Denaturation; D055503:Protein Multimerization; D020543:Proteome / Q000032:analysis*; D020027:Pyrococcus furiosus / Q000378:metabolism*
D000596:Amino Acids; D019843:Archaeal Proteins; D046912:Multiprotein Complexes; D020543:Proteome