Membrane Proteins Are Dramatically Less Conserved than Water-Soluble Proteins across the Tree of Life

Abstract

Membrane proteins are crucial in transport, signaling, bioenergetics, catalysis, and as drug targets. Here, we show that membrane proteins have dramatically fewer detectable orthologs than water-soluble proteins, less than half in most species analyzed. This sparse distribution could reflect rapid divergence or gene loss. We find that both mechanisms operate. First, membrane proteins evolve faster than water-soluble proteins, particularly in their exterior-facing portions. Second, we demonstrate that predicted ancestral membrane proteins are preferentially lost compared with water-soluble proteins in closely related species of archaea and bacteria. These patterns are consistent across the whole tree of life, and in each of the three domains of archaea, bacteria, and eukaryotes. Our findings point to a fundamental evolutionary principle: membrane proteins evolve faster due to stronger adaptive selection in changing environments, whereas cytosolic proteins are under more stringent purifying selection in the homeostatic interior of the cell. This effect should be strongest in prokaryotes, weaker in unicellular eukaryotes (with intracellular membranes), and weakest in multicellular eukaryotes (with extracellular homeostasis). We demonstrate that this is indeed the case. Similarly, we show that extracellular water-soluble proteins exhibit an even stronger pattern of low homology than membrane proteins. These striking differences in conservation of membrane proteins versus water-soluble proteins have important implications for evolution and medicine.

Keywords: adaptation; evolution; homeostasis; membrane proteins; orthologs.

Fig. 1

Two-fold effect of adaptation causes faster evolution of external sections and loss of homology in membrane proteins. Adaptation to new functions and niches causes faster evolution for outside-facing sections (top), potentially contributing to divergence beyond recognition. Other proteins may provide no advantage in the new environment, and could be lost entirely over time (center). For simplicity, the species on the left is assumed to remain functionally identical to the common ancestor (bottom).

Fig. 2

Membrane proteins have fewer orthologs in all three domains of life. The mean size of OMA Ortholog Groups (OGs) is substantially smaller for membrane proteins in all 64 species in the EMBL-EBI’s list of reference proteomes studied (2 of the 66 species were not found in OMA at the time of this analysis). Five-letter codes are OMA species identifiers; details in supplementary table S1, Supplementary Material online. Dark shade: water-soluble (WS); light shade: membrane proteins (MP). Data represented as the mean number of orthologs that WSs and MPs of each genome have in OMA±2×SEM (standard error of the mean).

Fig. 3

Water-soluble orthologous groups are substantially larger on average than membrane-protein groups, but the effect decreases as organismal complexity increases. Dividing the average size of water-soluble orthologous groups (OGs) of each species over the corresponding average size of membrane-protein OGs gives an indication of the magnitude of the effect in figure 2 for the different groups of species. (A) The ratio of the mean sizes of water-soluble over membrane protein OGs is > 1 for all species studied (i.e., each WS bar is always larger than its corresponding MP bar in figure 2), but the effect decreases as cellular and organismal complexity increase, from prokaryotes to unicellular eukaryotes, to multicellular eukaryotes. (B) Filtering for orthologous groups composed of both eukaryotes and prokaryotes keeps the relationship between unicellular and multicellular eukaryotes and indeed increases the effect, whereas prokaryotes remain largely unaltered. This suggests that membrane proteins ancestral to eukaryotes (i.e., with ancestors in archaea or bacteria) have been lost more often than their water-soluble counterparts. Bold black lines represent the median, white lines the mean, and boxes and whiskers are standard in R at a ± 1.5*IQR (inter-quartile range) threshold. Numbers below the boxes indicate sample sizes.

Fig. 4

The probability of a protein being membrane-bound falls with wider distribution. (A) A logistic regression shows that the probability that a gene is a membrane protein falls significantly with increasing number of clades sharing it, for OGs shared by any 3 or more separate clades. The pattern remains when considering each of the three domains separately (B–D). The points and vertical stripes correspond to the proportions of MPs amongst genes shared by increasingly large numbers of clades, divided in 10% bins. No proteins retrieved were shared by over 90% of the 489 taxa in (A). In all cases, the final bins have proportion zero, i.e., no highly shared proteins are membrane-bound. Note that the points and bins are provided for reference only: logistic regressions were performed on the individual ortholog clusters (i.e., the probability curves were derived independently, see Materials and Methods section).

Fig. 5

Membrane proteins evolve faster, especially in their external sections. (A–D) Nei’s sequence diversity measure (π) is higher for membrane proteins (MP) than for water-soluble proteins (WS) in the full set of OMA OGs (A) as well as for each of the three domains of life separately (B–D), indicating that evolution occurs faster for MPs. (E) For sections of membrane-protein OMA OGs annotated from the structures in the PDBTM database, Nei’s π shows that aqueous sections evolve faster overall than membrane-spanning sections. Splitting the aqueous sequences into outside- and inside-facing sections confirms that regions exposed to the environment evolve faster than those facing the cytosol. Boxplot ranges as in figure 3 with notches at the 95% confidence-interval around the median. All comparisons of WS to MP in (A–D), as well as inside and outside portions to each other or to membrane-spanning portions in (E) had P<0.001. Digits below the boxes indicate the numbers of orthologous groups.

Fig. 6

Extracellular proteins evolve faster and are shared by fewer species on average. (A) The mean ortholog cluster size is smaller for extracellular water-soluble than for membrane-bound proteins, whereas intracellular (cytosolic) proteins are shared by more species on average. (B) Binning proteins by the proportion of their amino-acid residues that are extracellular produces the mean OG sizes represented by the points, whereas the line is a simple linear regression on these points. See Materials and Methods section for details. (C) The evolutionary rates, estimated by Nei’s π, are higher for exported water-soluble proteins than for their intracellular counterparts, whereas membrane proteins show a slightly higher rate overall. Digits below the boxes indicate the numbers of orthologous groups.

Fig. 7

Ancestral membrane proteins have been lost more frequently. (A) Predicted ancestral proteins (defined as shared by at least half of the members of a clade), are shared by a smaller proportion of members in the clade if they are membrane proteins, for 31 of the 35 groups studied (exceptions are Neisseria, Rickettsia, Salmonella, and Yersinia). Dark shade: water-soluble; light shade: membrane proteins. First six pairs (blue) are archaeal clades, the remainder (yellow) are bacterial. Error bars: 2×SEM. Digits below bars indicate the numbers of orthologous groups. (B) Values as in (A), paired and without error bars. Red-dashed: results with higher mean proportion of sharing species for MP than WS. Yellow-dotted: results with MP<WS but P-value over a cutoff of 0.05 in a two-sample Welch t-test. Green-solid: results with P<0.05.

Fig. 8

An example of the alignment slicing process. Here, i, m, and o represent that the corresponding amino acid in the PDB structure is annotated as inside, membrane-spanning, or outside (respectively), either in the PDBTM database for figure 5E, or in the 12 annotations created by directly inspecting the PDB structure against the primary literature for supplementary figures S3 and S4, Supplementary Material online. In the example here, positions 1–2, and 15–19 are inside; 6–7 are outside; 3–5, 8–9, 25 and 12–14 are membrane-spanning; and 10–11, 20 are not present in the reference sequence and are therefore ignored.