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Supplemental Material 4: Subcellular Profiling of Plasmodium
falciparum Proteins Expressed in Sporozoites, Merozoites,
Trophozoites and Gametocytes.
From all four stages analysed, we identified 439 proteins known or predicted to
have at least one transmembrane segment or a GPI addition ‘signal’ at the Cterminus (18% of the dataset), 45% of which also contained an N-terminal leader
sequence. Our dataset also included 304 soluble proteins with a signal
sequence, i.e. proteins potentially secreted or located to organelles
(Supplementary Table 4). These structural predictions were based on the
TMHMM 1, big-PI Predictor 2 and SignalP 3-5 algorithms, which were run against
the entire Plasmodium falciparum genome. Well-characterized integral
membrane proteins (i.e, proton-pumping vacuolar pyrophosphatase, P-type
calcium-translocating ATPase, and nucleoside transporter 1) and secreted
proteins (i.e., KAHRP and CLAG) were identified. However, over half of the
potentially secreted and integral membrane proteins detected were annotated as
hypothetical (Supplementary Table 4). Cell surface proteins constituted the main
class of known proteins with one or two transmembrane domains, whereas
proteins with six or more transmembrane domains were mostly classified as
transport functions (Supplementary Table 4). Similarly, known proteins with a
signal peptide were mostly proteases and organellar proteins. By association,
some hypothetical proteins with signal peptides may be as yet uncharacterised
components of the apical organelles or cell surface.
To determine whether the predicted membrane associated proteins
specifically localize in membranes, we systematically sorted the proteins
identified from soluble and insoluble protein fractions, which were digested and
analysed independently (Supplementary Table 4). Compiling results from all four
stages, soluble and insoluble runs contributed 2,164 and 836 proteins,
respectively, 585 proteins being found in both (Supplementary Table 4). The
majority of these common proteins were abundant soluble proteins, such as
histones and ribosomal proteins, contaminating the membrane preparations
(Supplementary Table 4).
When comparing our experimental solubility-based fractionation results
with the computational predictions, it appeared that proteins known or predicted
to be secreted and/or integral to the membranes were found throughout the
soluble and insoluble fractions (Supplementary Figure 4a). Since no protease
inhibitors were used during the sample preparation, protein degradation due to
endogenous proteases can occur. Soluble peptides from integral membrane
proteins can be released and detected in soluble fractions. This would most likely
explain why many membrane proteins were identified from soluble fractions.
Whereas the membrane protein class constituted about 16 % of all proteins
detected in soluble fractions, 30% of the proteins uniquely identified from pellets
were predicted to be integral to the membrane (Supplementary Figure 4b),
suggesting that the insoluble fractions were enriched in membrane associated
proteins.
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a
1600
1400
Number of Proteins
1200
1000
800
600
400
200
0
Both
Supernatant only
Fractions
Pellet only
Number of Proteins
(% of Total Number of Proteins identified in Fraction)
b
SP = 0 and TM = 0
SP=1 and TM = 0
TM>= 1
100
90
80
70
60
50
40
30
20
10
0
Both
Supernatant only
Pellet only
Fractions
Supplementary Figure 4: Proteins identified in our analysis were sorted,
specifying the biochemical fractions in which they were detected. Three fractions
were defined: proteins identified in i) both soluble and insoluble analyses, ii)
supernatant only, or iii) membrane pellets only. For each fraction, 3 structural
classes were detailed: proteins known or predicted i) to be entirely soluble (SP =
0 and TM = 0), ii) to contain 1 signal peptide (SP=1 and TM =0) and iii) to have at
least 1 transmembrane segment or GPI modification site (TM>=1) (a). The
number of proteins in each structural class are plotted as a percentage of the
total number of proteins detected in the solubility-based fractions (b).
Interestingly, 144 proteins found uniquely in carbonate-extracted
membrane fractions did not display in their primary sequences any of the known
membrane interacting features (Supplementary Table 4). Among these were a
putative phospholipase and a phosphatidylserine decarboxylase (Supplementary
Table 4), which modify phospholipids, but which have no predicted
transmembrane component. An O-sialoglycoprotein endopeptidase, which
digests membrane-bound O-sialoglycoproteins, was detected uniquely in the
membrane fractions. Soluble subunits can also associate with transmembrane
complexes, such as the cytochrome c oxidase subunit II (Supplementary Table
4). Finally, proteins such as kinases and phosphatases, which were identified
from membrane fractions (Supplementary Table 4), associate and modify
membrane receptors as part of the signal transduction cascade. Of the 43 known
proteins found uniquely in membrane fractions, 26 have been shown to function
at the lipid interface (Supplementary Table 4).
Proteins involved in membrane functions are therefore not limited to
integral membrane proteins, soluble proteins are found at the lipid interface,
involved in many different processes from energy transduction to signalling.
Since it is problematic to identify proteins that associate with a lipid interface
based solely on primary amino acid sequence, the differential proteomic method
we describe here should help in the characterization of soluble proteins that
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appear to function at the lipid membrane. Taken together, our results indicate
that the soluble proteins found uniquely in membrane fractions, 101 of which are
hypothetical, can be inferred to have membrane-associated functions
(Supplementary Table 4).
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