Identification of membrane proteins in the hyperthermophilic archaeon Pyrococcus furiosus using proteomics and prediction programs

James F Holden,John R Yates Iii,Michael W W Adams,Farris L Poole Ii,Hanjo Lim,Carol S Giometti,Sandra L Tollaksen

doi:10.1002/cfg.110

Abstract

Cell-free extracts from the hyperthermophilic archaeon Pyrococcus furiosus were separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning α-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanning β-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80%. A consensus prediction using all six programs for all 2242 ORFs in the P. furiosus genome estimates that 24% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs’ inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

Highlights

The advent of genome sequencing has revolutionized the study of microbial physiology
Membrane and cytoplasmic fractions were prepared from P. furiosus cells grown on 0.5% (w/v) each of maltose, yeast extract, and casein hydrolysate with and without Su and from cells grown on 0.5% maltose and 0.05% yeast extract without Su
Our results indicate that between 7 and 17% of the total cellular protein is located in the membrane fraction of P. furiosus, depending on the growth conditions

Summary

Introduction

The advent of genome sequencing has revolutionized the study of microbial physiology. Single protein characterizations and biochemical pathway studies have been augmented by our ability to determine the functional relationships between different pathways and the roles of novel proteins This approach, known as functional genomics, typically involves the use of DNA microarrays and proteomics (Dove, 1999; Southern et al, 1999). The location of proteins can be determined physically by separating cell-free extracts into cytoplasmic and membrane-associated fractions and by assessing the protein species present in each. This typically involves twodimensional electrophoresis and the identification of proteins using mass spectrometry (Cordwell et al, 2000). Separation of membrane and cytoplasmic proteins prior to proteomic analyses considerably improved the resolution and ease of identification of membrane proteins from the bacterium Pseudomonas aeruginosa PAO1 (Nouwens et al, 2000)

Methods

Results

Conclusion