<p>This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</p> <p>Cell-free extracts from the hyperthermophilic archaeon <italic>Pyrococcus furiosus</italic> 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 <italic>β</italic>-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 <italic>P. furiosus</italic> 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.</p>
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Comp Funct Genomics. 2001 Oct; 2(5):275-288