Size Comparisons among Integral Membrane Transport Protein Homologues in Bacteria, Archaea, and Eucarya

JOURNAL OF BACTERIOLOGY, Feb. 2001, p. 1012–1021 0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.3.1012–1021.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Vol. 183, No. 3

Size Comparisons among Integral Membrane Transport Protein Homologues in Bacteria, Archaea, and Eucarya YONG JOON CHUNG,† CHRISTEL KRUEGER, DAVID METZGAR,

AND

MILTON H. SAIER, JR.*

Department of Biology, University of California at San Diego, La Jolla, California 92093-0116 Received 7 July 2000/Accepted 3 November 2000

Integral membrane proteins from over 20 ubiquitous families of channels, secondary carriers, and primary active transporters were analyzed for average size differences between homologues from the three domains of life: Bacteria, Archaea, and Eucarya. The results showed that while eucaryotic homologues are consistently larger than their bacterial counterparts, archaeal homologues are significantly smaller. These size differences proved to be due primarily to variations in the sizes of hydrophilic domains localized to the N termini, the C termini, or specific loops between transmembrane ␣-helical spanners, depending on the family. Within the Eucarya domain, plant homologues proved to be substantially smaller than their animal and fungal counterparts. By contrast, extracytoplasmic receptors of ABC-type uptake systems in Archaea proved to be larger on average than those of their bacterial homologues, while cytoplasmic enzymes from different organisms exhibited little or no significant size differences. These observations presumably reflect evolutionary pressure and molecular mechanisms that must have been operative since these groups of organisms diverged from each other. tently larger than their bacterial counterparts, the archaeal homologues are almost always smaller. Moreover, within the Eucarya domain, plant homologues are consistently smaller than the fungal and animal homologues, which are of similar sizes. These observations apparently do not apply to extracellular receptors and cytoplasmic enzymes, which exhibit the reverse size tendencies or no significant differences. The size differences observed for secondary carriers of homologues from the three domains of life proved to be due primarily to variations in the sizes of specific hydrophilic domains within these proteins, and the locations of these size-variable domains appear to be characteristic of specific families.

The three largest classes of transporters found in nature are channels, secondary carriers, and primary active transporters (8, 10). Channel proteins facilitate passive diffusion of their substrates across membranes through aqueous pores, while secondary carriers generally utilize electrochemical gradients of H⫹, Na⫹, and solutes to drive the active accumulation or efflux of their primary substrates, and primary active transporters couple transport to the expenditure of a primary source of energy such as ATP hydrolysis or electron flow (10, 16). While channel proteins frequently span the membrane only a few times and form oligomeric complexes, secondary carriers and primary active transporters span the membrane multiple times and usually function as monomers or dimers in the absence of accessory proteins (4). Higher complexes of primary and secondary active transporters can provide regulatory (7, 11) or targeting and stability functions (15). Recently we have classified transport proteins according to a functional and phylogenetic system called the transporter classification (TC) system (8–10). While many of the identified families of transport proteins are found in only one of the three domains of living organisms (Bacteria, Archaea, or Eucarya), others are ubiquitous, being found in all three domains. Our studies have led to the conclusion that these ubiquitous families are ancient families that existed prior to the divergence of Eucarya and Archaea from Bacteria and that little horizontal transfer of genetic material encoding transport proteins between these three domains of life has occurred at least during the past 2 to 3 billion years (8, 9). In this study we compared the sizes of homologues of the ubiquitous families in the three domains of living organisms. We showed that while the eucaryotic homologues are consis-

MATERIALS AND METHODS The PSI-BLAST database search method (http://www.ncbi.nlm.nih.gov/blast /psiblast.cgi) was used to identify homologous proteins. Multiple alignments were generated using the CLUSTAL X program (13), and hydropathy and putative transmembrane spanner (TMS) analyses were conducted using the TMPred program (2). Positions of size variation among homologues were identified using a combination of programs for multiple alignment (CLUSTAL X) and topological analysis (TMPred). To test for statistically significant differences in protein length, the data were analyzed using two-tailed Sign tests (17).

RESULTS Size variation in integral membrane transport protein homologues in Bacteria, Archaea, and Eucarya. Table 1 presents the average sizes, in numbers of amino acyl residues, of the integral membrane protein homologues of 15 families of secondary carriers, 3 families of channel proteins, and 4 families of primary active transporters present in the archaeal, bacterial, and eucaryotic domains. The number of homologues examined is presented in parentheses. The average sizes of the archaeal and eucaryotic homologues relative to the average sizes of the bacterial proteins are also provided. All of the archaeal homologues available in the SwissProt, GenBank, and PIR databases at the time these studies were conducted were included in the analysis. When limited numbers of bacterial or eucaryotic homologues comparable to the number of archaeal

* Corresponding author. Mailing address: Department of Biology, University of California at San Diego, La Jolla, CA 92093-0116. Phone: (858) 534-4084. Fax: (858) 534-7108. E-mail: msaier@ucsd .edu. † Permanent address: Department of Life Science, Jeonju University, Chonju, Korea. 1012

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TABLE 1. Comparison of membrane transport homologue sizes between Archaea, Bacteria, and Eucarya Avg size Family

Archaea

TC no.a

Bacteria

Eucarya

No. of AAsb

% Relative sizec

No. of AAsb

No. of AAsb

% Relative sizec

124 131 165 130

Carriers Sugar porter (major facilitator superfamily) Amino acid-polyamine-organocation Cation diffusion facilitator Resistance-nodulation-division SecDF Ca2⫹:cation antiporter Inorganic phosphate transporter Monovalent cation:proton antiporter-1 Monovalent cation:proton antiporter-2 K⫹ transporter Nucleobase:cation symporter-2 Formate-nitrite transporter Divalent anion:Na⫹ symporter Ammonium transporter Multi-antimicrobial extrusion

2.A.1.1 2.A.3 2.A.4 2.A.6 2.A.6.4 2.A.19 2.A.20 2.A.36 2.A.37 2.A.38 2.A.40 2.A.44 2.A.47 2.A.49 2.A.66

399 (4) 508 (6) 293 (4) 758 (3) 713 (3) 320 (4) 332 (6) 440 (3) 410 (5) 477 (3) 439 (3) 277 (2) 410 (4) 411 (7) 454 (5)

95 109 98 76 76 87 70 85 84 99 97 102 79 88 99

422 (6) 463 (6) 298 (4) 998 (56) 935 (13) 370 (7) 477 (5) 516 (5) 490 (13) 480 (5) 452 (9) 273 (7) 516 (8) 464 (7) 458 (5)

527 (18) 602 (5) 491 (8) 1,296 (4) —d 649 (24) 581 (10) 702 (15) 793 (5) 758 (8) 566 (13) 547 (2) 681 (12) 503 (12) 636 (6)

174 122 136 162 157 125 200 132 108 138

Channels Major intrinsic protein Chloride channel Metal ion transporter

1.A.8 1.A.11 9.A.17

246 (2) 410 (5) 330 (3)

98 89 99

251 (11) 458 (5) 332 (19)

278 (33) 827 (17) 692 (9)

111 180 210

3.A.3 3.A.4 3.A.5 3.B.1

724 (9) 388 (7) 461 (12) 385 (3)

99 94 106 96

732 (85) 411 (22) 436 (44) 399 (9)

1,096 (62) 693 (11) 455 (26) —d

150 169 104

Primary active transporters P-type ATPase Arsenite-antimonite efflux Type II secretory pathway (SecY) Na⫹-transporting carboxylic acid decarboxylase (␤) a

For further information on TC numbers, see reference 10 or http://www-biology.ucsd.edu/⬃msaier/transport/. The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses. Average percent size of the archaeal or eukaryotic homologue relative to the bacterial homologue is presented for each of the families examined. When the values for all families were averaged, the archaeal proteins proved to be 8% smaller than their bacterial homologues while the eukaryotic homologues proved to be 40% larger. d No protein homologues were identified in this domain. b c

proteins were identified, all of these were also included. However, when the numbers of bacterial and/or eucaryotic homologues considerably exceeded the number of archaeal family members, several proteins from the former two groups were generally selected at random from various organisms. In some cases, many eucaryotic proteins were included so that proteins within specific Eucarya kingdoms (animals, plants, and fungi) could be compared (see below). Examination of the results presented in Table 1 reveals that of the 22 protein families studied, the average sizes of the eucaryotic homologues are always substantially greater than those of the procaryotic homologues. Moreover, with only three exceptions (the amino acid-polyamine-organocation [APC] and formate-nitrite transporter [FNT] families of secondary carriers and the SecY proteins of the type II protein secretion pathway family of primary active protein secretory systems), the average sizes of the archaeal homologues are always less than those of the bacterial homologues. All of the size difference values, obtained when the archaeal or eucaryotic homologues for the various families were compared with the bacterial homologues (Table 1), were averaged. The average archaeal protein size for all 22 families examined was 92% of that of the bacterial homologues, while the average eucaryotic protein size for all 20 families examined was 140%

of that of the bacterial homologues. Thus, while the archaeal proteins are 8% smaller than the bacterial proteins, on average, the eucaryotic proteins are 40% larger. Size variation in integral membrane transport protein homologues in fungi, plants, and animals. Within the Eucarya domain, animal, plant, and fungal (including yeast) homologues were analyzed separately (Table 2). In all but three of the families of transport proteins analyzed, the plant proteins exhibited average sizes that were substantially smaller than the animal or fungal homologues. The exceptions were the sugar porter family of the major facilitator superfamily, the ammonium transporter family, and the SecY family within type II protein secretion pathway systems. In the sugar porter family of the major facilitator super family, animal homologues proved to be slightly smaller on average than the plant homologues. All of the size difference values, obtained when the animal or plant homologues for the various families were compared with the fungal homologues (Table 2), were averaged. The average animal protein size for all 14 families examined was 105% of that of the fungal homologues, while the average plant protein size for the 13 families examined was 83% of that of the fungal homologues. Thus, while the animal proteins are

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TABLE 2. Comparison of membrane transport homologue sizes between animals, plants, and fungi Avg size Family

Animals

TC no.

Plants

Fungi

No. of AAsa

% Relative sizeb

No. of AAsa

% Relative sizeb

No. of AAsa

91 85 72 90 74 89 50 109

571 (6) 557 (9) 610 (6) 1,085 (2) 657 (3) 588 (2) 1,010 (6) 891 (4) 452 (3)

Carriers Sugar porter (major facilitator superfamily) Amino acid/auxin porter Ca2⫹:cation antiporter Cation-chloride cotransporter Monovalent cation:proton antiporter-1 Nucleobase:cation symporter-2 K⫹ transporter Divalent anion:Na⫹ symporter Ammonium transporter

2.A.1.1 2.A.18 2.A.19 2.A.30 2.A.36 2.A.40 2.A.43 2.A.47 2.A.49

491 (6) 517 (13) 897 (14) 1,060 (14) 755 (10) 591 (6) —c 575 (8) 576 (4)

86 93 147 98 115 101 65 127

518 (6) 475 (16) 441 (4) 973 (1) 488 (2) 523 (5) 506 (2) —c 493 (5)

Channels Major intrinsic protein Chloride channel Metal ion transporter

1.A.8 1.A.11 9.A.17

315 (15) 858 (11) 725 (3)

102 108 102

268 (22) 778 (6) 657 (4)

87 98 93

308 (2) 796 (2) 710 (2)

Primary active transporters P-type ATPase Arsenite-antimonite efflux Type II secretory pathway (SecY)

3.A.3 3.A.4 3.A.5

1,348 (21) 667 (9) 440 (6)

135 82 113

954 (29) —c 471 (16)

95

999 (12) 809 (2) 391 (3)

120

a

The average number of amino acyl residues (AAs) for the protein homologues of each family is reported, with the number of proteins examined in parentheses. Fungal proteins include those from yeast. b Average percent size of the animal or plant homologue relative to the fungal homologue is presented for each of the families examined. When the values for all families were averaged, the plant proteins proved to be 17% smaller than their fungal homologues while the animal homologues proved to be 5% larger. c No protein homologues were identified in this kingdom.

5% larger than the fungal proteins, on average, the plant proteins are 17% smaller. Size variation in homologous constituents of the ABC-type transport system in Bacteria and Archaea. The ABC superfamily of uptake permeases is restricted to procaryotes, but it is found in both Bacteria and Archaea. These systems include three constituents: extracytoplasmic receptors, integral membrane proteins, and cytoplasmic ATP-hydrolyzing constituents. Over 20 families of these systems have been identified (10). These types of homologues (receptors, integral membrane constituents, and cytoplasmic ATP-hydrolyzing energizers) were analyzed for size variation (see Tables 3, 4, and 5, respectively). As shown in Table 3, the average archaeal receptor sizes proved to be greater than those of the average bacterial receptor sizes for 11 of the 13 families that have homologues in both domains. Overall, the archaeal receptors are 7% larger, on average, than their bacterial homologues. By contrast, the integral membrane archaeal homologues of ABC systems are usually smaller than the bacterial homologues (Table 4). Thus, of the 20 families examined, 15 proved to have smaller archaeal homologues, on average, than bacterial homologues. The average size difference proved to be 3.5%. Finally, the cytoplasmic ATP-hydrolyzing energizers tend to be somewhat smaller in Archaea than in Bacteria (Table 5). Thus, of the 16 families analyzed, 13 were smaller and 3 were larger, on average. Overall, the archaeal cytoplasmic proteins were 3.5% smaller than their bacterial homologues. Thus, the trend displayed by the ABC membrane proteins (Table 4) agreed with that for other integral membrane transport proteins (Table 1). The size differences for the archaeal extracytoplasmic receptors were opposite to that observed for the integral membrane constituents, with the archaeal receptors being substantially

larger than their bacterial homologues. The ATP-hydrolyzing energizers showed minimal size differences. Size variation in homologous cytoplasmic enzymes. Similar analyses were conducted with a variety of catabolic and anabolic cytoplasmic enzymes (Table 6). These proteins showed similar homologue sizes, regardless of the domain or kingdom analyzed. Averaging all of the statistically significant results in Table 6 revealed that, on average, eucaryotic enzymes are only 3% larger than the homologous bacterial enzymes and archaeal enzymes are only 3% smaller than the homologous bacterial enzymes. These average size differences are much less than for the integral membrane transport proteins analyzed (Tables 1 and 4). Moreover, among the Eucarya kingdoms, animal and fungal homologues are essentially the same size while plant homologues are only about 1% smaller on average. This last mentioned average size difference is not statistically significant. Thus, cytoplasmic enzymes do not appear to exhibit the appreciable size differences that were observed for integral membrane proteins. Statistical significance of the observed homologue size differences. To test for statistically significant differences in transport protein lengths between phylogenetic groups, the data were analyzed using two-tailed Sign tests (17). In these analyses, comparisons were made between paired domains of life, or between paired kingdoms within the Eucarya domain, in terms of average lengths of amino acyl sequences within the protein families (Tables 1 to 5). The Sign test is a qualitative, nonparametric paired-sample test that utilizes only the direction of difference (⬍ or ⬎) between paired data. As such, it requires no assumptions regarding the distribution of data either within or between sample groups. We felt that such assumptions might be unwarranted given the size variation observed within

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TABLE 3. Comparison of ABC receptor homologue sizes between Archaea and Bacteria Avg size Family

Carbohydrate uptake transporter-1 family Carbohydrate uptake transporter-2 family Polar amino acid uptake transporter family Hydrophobic amino acid uptake transporter family Peptide-opine-nickel uptake transporter family Sulfate uptake transporter family Phosphate uptake transporter family Molybdate uptake transporter family Phosphonate uptake transporter family Ferric iron uptake transporter family Polyamine-opine-phosphonate uptake transporter family Quaternary amine uptake transporter family Vitamin B12 uptake transporter family Iron chelate uptake transporter family Manganese-zinc-iron chelate uptake transporter family Nitrate-nitrite-cyanate uptake transporter family Taurine uptake transporter family Putative cobalt uptake transporter family Thiamine uptake transporter family Brachyspira iron transporter family

Archaea

TC no.

Bacteria

No. of AAsa

% Relative sizeb

No. of AAsa

% Relative sizeb

477 (6) —c 278 (2) 443 (4) 644 (11) —c 337 (6) 264 (2) —c —c 417 (2) —c 361 (4) 347 (2) 316 (4) 353 (4) —c 88 (4) 350 (2) —c

111

421 (13) 343 (12) 262 (29) 376 (14) 532 (25) 342 (6) 334 (18) 251 (15) 300 (5) 328 (15) 352 (20) 421 (7) 279 (14) 327 (20) 306 (26) 451 (10) 333 (11) 105 (3) 346 (12) 346 (10)

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

3.A.1.1 3.A.1.2 3.A.1.3 3.A.1.4 3.A.1.5 3.A.1.6 3.A.1.7 3.A.1.8 3.A.1.9 3.A.1.10 3.A.1.11 3.A.1.12 3.A.1.13 3.A.1.14 3.A.1.15 3.A.1.16 3.A.1.17 3.A.1.18 3.A.1.19 3.A.1.20

106 118 121 101 105 118 129 106 103 78 84 101

a

The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses. Average percent size of the archaeal homologues relative to the bacterial homologues is presented for each of the families examined. c No protein homologue or just one such protein was identified in this domain. b

the taxonomic groups with respect to average length of the proteins within the families represented. Thus, there proved to be more variation between protein families within each domain than there was between domains in any particular protein family. Each pair of domains or kingdoms was therefore com-

pared independently. A P value of ⱕ0.05 was considered significant. Tabulated data and associated Sign test P values are presented in Table 7. The results of the statistical analyses strongly support the conclusion that transport protein length differs significantly

TABLE 4. Comparison of ABC membrane protein homologue sizes between Archaea and Bacteria Avg size Family

Archaea

TC no. No. of AAs

Carbohydrate uptake transporter-1 family (MalF) Carbohydrate uptake transporter-1 family (MalG) Carbohydrate uptake transporter-2 family (RbsC) Carbohydrate uptake transporter-2 family (RbsD) Polar amino acid uptake transporter family (HisM) Polar amino acid uptake transporter family (HisQ) Hydrophobic amino acid uptake transporter family (LivH) Hydrophobic amino acid uptake transporter family (LivM) Peptide-opine-nickel uptake transporter family (OppB) Peptide-opine-nickel uptake transporter family (OppC) Sulfate uptake transporter family Phosphate uptake transporter family Molybdate uptake transporter family Phosphonate uptake transporter family Ferric iron uptake transporter family Polyamine-opine-phosphonate uptake transporter family (PotB) Polyamine-opine-phosphonate uptake transporter family (PotC) Quaternary amine uptake transporter family Vitamin B12 uptake transporter family Iron chelate uptake transporter family (FecC) Iron chelate uptake transporter family (FecD) Manganese-zinc-iron chelate uptake transporter family Nitrate-nitrite-cyanate uptake transporter family Taurine uptake transporter family Putative cobalt uptake transporter family Thiamine uptake transporter family Brachyspira iron transporter family a b c

3.A.1.1 3.A.1.1 3.A.1.2 3.A.1.2 3.A.1.3 3.A.1.3 3.A.1.4 3.A.1.4 3.A.1.5 3.A.1.5 3.A.1.6 3.A.1.7 3.A.1.8 3.A.1.9 3.A.1.10 3.A.1.11 3.A.1.11 3.A.1.12 3.A.1.13 3.A.1.14 3.A.1.14 3.A.1.15 3.A.1.16 3.A.1.17 3.A.1.18 3.A.1.19 3.A.1.20

301 (4) 309 (4) 332 (1) —c 222 (2) 222 (2) 289 (5) 351 (8) 333 (12) 366 (9) —c 285 (7) 248 (4) —c —c 263 (3) 262 (3) —c 345 (6) 338 (6) 344 (4) 271 (4) 254 (5) —c 255 (30) 406 (4) —c

a

Bacteria

% Relative size

83 96 97 96 95 91 87 103 116 90 107 90 97 101 99 99 93 83 89 117

b

No. of AAs

363 (24) 321 (24) 342 (22) 137 (7) 232 (50) 233 (39) 318 (17) 402 (20) 324 (48) 315 (51) 277 (10) 315 (23) 232 (13) 246 (5) 520 (8) 291 (25) 271 (18) 435 (7) 341 (11) 340 (18) 346 (22) 291 (38) 305 (16) 328 (7) 287 (11) 348 (31) 424 (7)

a

% Relative sizeb

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses. Average percent size of the archaeal homologues relative to the bacterial homologues is presented for each of the families examined. No protein homologues were identified in this domain.

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TABLE 5. Comparison of cytoplasmic ABC protein homologue sizes between Archaea and Bacteria Avg size Family

Carbohydrate uptake transporter-1 family Carbohydrate uptake transporter-2 family Polar amino acid uptake transporter family Hydrophobic amino acid uptake transporter family Peptide-opine-nickel uptake transporter family Sulfate uptake transporter family Phosphate uptake transporter family Molybdate uptake transporter family Phosphonate uptake transporter family Ferric iron uptake transporter family Polyamine-opine-phosphonate uptake transporter family Quaternary amine uptake transporter family Vitamin B12 uptake transporter family Iron chelate uptake transporter family Manganese-zinc-iron chelate uptake transporter family Nitrate-nitrite-cyanate uptake transporter family Taurine uptake transporter family Putative cobalt uptake transporter family Thiamine uptake transporter family Brachyspira iron transporter family a b c

Archaea

TC no.

3.A.1.1 3.A.1.2 3.A.1.3 3.A.1.4 3.A.1.5 3.A.1.6 3.A.1.7 3.A.1.8 3.A.1.9 3.A.1.10 3.A.1.11 3.A.1.12 3.A.1.13 3.A.1.14 3.A.1.15 3.A.1.16 3.A.1.17 3.A.1.18 3.A.1.19 3.A.1.20

Bacteria

No. of AAsa

% Relative sizeb

No. of AAsa

% Relative sizeb

363 (14) 496 (5) 240 (2) 258 (6) 324 (9) 329 (1) 286 (10) 344 (1) —c 329 (1) 343 (5) 369 (3) 252 (3) 272 (2) 271 (3) 250 (3) —c 284 (9) —c —c

99 98 95 97 96 92 92 99

366 (48) 504 (34) 252 (31) 267 (15) 336 (25) 357 (8) 312 (21) 349 (8) 287 (4) 353 (9) 376 (13) 396 (12) 263 (10) 269 (24) 262 (21) 274 (18) 332 (11) 276 (11) 225 (3) 371 (3)

100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100

93 91 93 96 101 103 91 103

The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses. Average percent size of the archaeal homologue relative to the bacterial homologue is presented for each of the families examined. No protein homologues were identified in this domain.

between domains in all pairwise comparisons. Archaea have significantly shorter transport proteins than Bacteria, and both Archaea and Bacteria have shorter transport proteins than Eucarya. Tests were generally less significant in pairwise comparisons of Eucarya kingdoms. Plants have shorter transport proteins than either animals or fungi, but the difference in protein length between animals and fungi is not significant. Corresponding analyses of the ABC receptors, membrane proteins, and cytoplasmic energizers argued for statistical significance, although the actual size differences between the archaeal and bacterial energizers proved to be minimal. Localization of regions in homologues of secondary transporters responsible for size differences between Bacteria, Archaea, and Eucarya. Five families of secondary carriers were analyzed in detail to determine what portions of these proteins exhibit the greatest size variation. For this purpose, five sequence-divergent members of each family from each of the three domains of living organisms were selected for analysis.

These sequences were multiply aligned using the CLUSTAL X program (13), and hydropathy analyses were conducted using the TMpred program (2). The results of these analyses are summarized in Table 8. For each family, the bacterial homologues are presented first, the archaeal homologues are presented second, and the eucaryotic proteins are presented last. Table 8 presents (i) the organismal domains, (ii) the protein abbreviations, (iii) the size of each individual protein, (iv) the database and accession number, allowing easy access to the sequence of that protein, (v) the number of putative TMSs predicted using the TMpred program, (vi) the size of the N-terminal hydrophilic domain (N) in number of amino acyl residues, (vii) the residues predicted to comprise the individual TMSs (1 to 14), and (viii) the size of the C-terminal hydrophilic domain (C). The first family shown is the Ca2⫹:cation antiporter (CaCA) family (Table 8). The size differences between the proteins are apparent when examining the data summarized in column 3. In

TABLE 6. Comparisons of cytoplasmic enzyme sizes for the domains of organisms and the kingdoms of Eucarya Enzyme

Enolase Phosphoglycerate kinase Glyceraldehyde 3-phosphate dehydrogenase Triose-phosphate isomerase Phosphoglucose isomerase Pyruvate kinase Inosine-5⬘-monophosphate dehydrogenase Inosine-5⬘-monophosphate-aspartate ligase Glutamine synthetase Aspartate aminotransferase Elongation factor-2/elongation factor-G a

EC no.

Archaea

Bacteria

4.2.1.11 2.7.2.3 1.2.1.12 5.3.1.1 5.3.1.9 2.1.7.40 1.1.1.250 6.3.4.4 6.3.1.2 2.6.1.1 No EC no.

425 (6) 409 (10) 338 (12) 224 (10) 401 (1) 465 (4) 481 (7) 340 (6) 451 (11) 391 (6) 731 (16)

430 (23) 398 (21) 335 (24) 253 (25) 517 (22) 500 (22) 500 (16) 426 (19) 466 (26) 394 (17) 698 (25)

No sequenced homologues of this enzyme were found in plants.

Eucarya All kingdoms

Animals

Plants

Fungi

438 (30) 414 (27) 336 (57) 251 (24) 560 (18) 522 (22) 517 (11) 447 (6) 369 (33) 412 (15) 849 (12)

434 (13) 416 (11) 334 (23) 249 (11) 557 (7) 529 (9) 520 (4) 454 (4) 376 (13) 412 (8) 855 (6)

444 (8) 401 (4) 339 (13) 254 (8) 566 (8) 509 (6) 502 (3) —a 365 (15) 409 (5) 845 (3)

438 (9) 417 (12) 335 (21) 249 (5) 554 (3) 524 (7) 525 (4) 434 (2) 360 (5) 421 (2) 842 (3)

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TABLE 7. Results of statistical analyses of observed homologue size differences Homologue

Groups compared

No. with A ⬎ B:A ⬍ B

Sign test P value

Correlation

Integral membrane transporter

Archaea (A) vs Bacteria (B) Bacteria (A) vs Eucarya (B) Eucarya (A) vs Archaea (B) Plants (A) vs animals (B) Animals (A) vs fungi (B) Fungi (A) vs plants (B) Archaea (A) vs Bacteria (B) Archaea (A) vs Bacteria (B) Archaea (A) vs Bacteria (B)

3:19 0:20 19:1 2:10 9:5 11:2 11:2 5:15 3:13

0.002 ⬍0.001 ⬍0.001 0.039 0.42 0.022 0.022 0.041 0.013

A⬍B A⬍B A⬎B A⬍B None A⬎B A⬎B A⬍B A⬍B

ABC receptor ABC membrane protein ABC cytoplasmic energizer

column 5, it can be seen that there is substantial variation in the predicted number of TMSs. For this family and other families examined, some of this variation may represent experimental error due to limitations of the TMPred program. For the CaCA family, there is little variation in the sizes of the N-terminal and C-terminal hydrophilic domains (between 0 and 40 residues each). However, three of the eucaryotic proteins (all from animals) (CaSA2 Bta, Orfl Cel, and CaSA Dme) show large inter-TMS loops between TMSs 6 and 7. These loops are between 455 and 566 amino acyl residues long, accounting for most of the size differences observed for these proteins compared with other homologues examined. These loops are predicted to be of 29 to 38 residues for the bacterial proteins, of 11 to 22 residues for the archaeal proteins, and of 14 and 10 residues for the two remaining eucaryotic proteins, plant and yeast proteins, respectively. Additionally, it can be seen that other eucaryotic inter-TMS loops are of somewhat increased size relative to their procaryotic counterparts. For example, loops 1 and 2 (between TMSs 1 and 2) contain 1 to 22 residues in procaryotic proteins but 34 to 99 residues in the eucaryotic homologues; loops 2 and 3 in the procaryotic proteins are 16 to 19 residues long while those in the eucaryotic proteins are 21 to 23 residues long; and loops 3 and 4 in the procaryotic proteins are of 7 to 18 residues while those in the eucaryotic proteins are of 15 to 23 residues. Finally, the program predicts 11 TMSs for the bacterial homologues, 9 or 10 TMSs for the archaeal homologues, and either 10 or 12 TMSs for the eucaryotic proteins. All of these differences, when taken together, account for the observed size variations of the individual proteins of the CaCA family. The second family listed in Table 8 is the inorganic phosphate transporter (Pit) family. All but one of the Pit family members has a small N-terminal hydrophilic region, the one exception being the plant Pit Ath homologue, which has an N-terminal hydrophilic domain of 126 residues. The archaeal proteins generally have shorter hydrophilic N termini than the bacterial proteins. Further, the hydrophilic C termini of all homologues are short (1 to 26 residues). The major size variations observed between the procaryotic and eucaryotic proteins of this family are in loops 7–8 and 8–9. For example, Orf Cel is predicted to have a somewhat large loop 8–9 (46 residues), Glvr Hsa and Nps Sce have a large loop 7–8 (62 and 206 residues, respectively), and Pho4 Ncr has large loops 7–8 and 8–9 (122 and 89 residues, respectively). Differences in the number of putative TMSs predicted are also observed, with

Orf Cel and Pho4 Ncr predicted to have more TMSs than the other homologues. The monovalent cation:proton antiporter families (CPA1 and CPA2) show similarly sized N-terminal hydrophilic domains, but their C-terminal hydrophilic domains differ substantially in size (Table 8). Thus, in the CPA1 family, four of the bacterial homologues have hydrophilic extensions of 21 to 32 residues, but one protein, Orf Bsu, has a hydrophilic extension of 131 residues. Similarly, four of the archaeal proteins have C-terminal hydrophilic extensions of 7 to 25 residues, but one protein (Nhe2 Afu) has an extension of 124 residues. Finally, all of the eucaryotic proteins have long C-terminal hydrophilic domains of 102 to 394 residues. In the CPA2 family, the major size differences are also in the C-terminal regions. In this family, the bacterial C-terminal extensions are large (226 to 282 residues), while all but one of the archaeal extensions are short (6 to 21 residues except for that in Orf Mth, which is 174 residues in length). All of the eucaryotic proteins have large hydrophilic C termini (185 to 277 residues). The two yeast proteins additionally exhibit large loops between their final two C-terminal TMSs (308 and 444 residues, respectively). Finally, several proteins of the divalent anion:Na⫹ symporter family exhibit major size differences in their N-terminal hydrophilic domains (Table 8), although differences in loop sizes and numbers of putative TMSs contribute significantly to the overall protein size differences. Most of these proteins show small C-terminal hydrophilic extensions. In summary, we have found that the positional basis for the size variations observed between secondary carrier homologues from the three domains of life depends primarily on the family and secondarily on the individual proteins within that family. Some families show differences primarily in the Nterminal hydrophilic domains, others show differences in the C-terminal hydrophilic domains, and still others show differences in specific inter-TMS loop regions. Most families exhibit size differences between homologues that represent a combination of these effects, with one of these effects predominating. DISCUSSION The average size differences for the various types of protein homologues analyzed are summarized in Table 9. When all family size differences are averaged, integral membrane transport proteins of bacteria are 8% larger than their archaeal homologues and 40% smaller than their eucaryotic homo-

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TABLE 8. Localization of regions of size difference between homologues of five families of secondary carriers Homologue family (TC)

Ca2⫹:cation antiporter (2.A.19)

Group

Name

Bacteria

ChaA Eco CaHA Syn Orf21 Ype ChaA Mtu Y4HA Rhi Orf1 Mth Orf2 Mth Orf Mja Orf Pho Orf Pab CaSA2 Bta Orf1 Cel CaSA Dme CAX1 Ath Orf3 Sce PitA Eco YG04 Hin PitB Mtu Orf Cje Orf Cpn Y630 Mja Orf Afu Orf Mth Orf Pho Orf Pab Orf Cel Glvr Hsa Pit Ath Pho4 Ncr Npa Sce YjcE Eco Orf Syn Orf Mtu NhaP Pae Orf Bsu Nhe2 Afu Orf Mth Y057 Mja Orf Pab Orf Pho Orf Cel NahB Cmy Nhel Hsa Nhx1 Ath Orf Spo KefC Eco KefC Mxa KefB Vch Orf Pae Orf Nme Orf Mth PhaC Mex Orf Pab Orf Pho Orf Mja Orf1 Ath Orf2 Ath Orf3 Ath Orf Sce Orf Spo Orf Reu Orf Hin YbdS Eco Orf Syn Orf Nme Orf Mja Orf Mth Orf Afu Orf Pho Orf Ape NadC Hsa Orf Cel Orf Ath Orf Sce Orf Spo

Archaea

Eucarya

Inorganic phosphate transporter (2.A.20)

Bacteria

Archaea

Eucarya

Monovalent cation:proton antiporter-1 (2.A.36)

Bacteria

Archaea

Eucarya

Monovalent cation:proton antiporter-2 (2.A.37)

Bacteria

Archaea

Eucarya

Divalent anion:Na⫹ symporter (2.A.47)

Bacteria

Archaea

Eucarya

a

Organism

Escherichia coli Synechocystis sp. Yersinia pestis Mycobacterium tuberculosis Rhizobium sp. Methanobacterium thermoautotrophicum Methanobacterium thermoautotrophicum Methanococcus jannaschii Pyrococcus horikoshii Pyrococcus abyssi Bos taurus Caenorhabditis elegans Drosophila melanogaster Arabidopsis thaliana Saccharomyces cerevisiae Escherichia coli Haemophilus influenzae Mycobacterium tuberculosis Campylobacter jejuni Chlamydophila pneumoniae Methanococcus jannaschii Archaeoglobus fulgidus Methanobacterium thermoautotrophicum Pyrococcus horikoshii Pyrococcus abyssi Caenorhabditis elegans Homo sapiens Arabidopsis thaliana Neurospora crassa Saccharomyces cerevisiae Escherichia coli Synechocystis sp. Mycobacterium tuberculosis Pseudomonas aeroginosa Bacillus subtilis Archaeoglobus fulgidus Methanobacterium thermoautotrophicum Methanococcus jannaschii Pyrococcus abyssi Pyrococcus horikoshii Caenorhabditis elegans Oncorhynchus mykiss Homo sapiens Arabidopsis thaliana Schizosaccharomyces pombe Escherichia coli Mixococcus xanthus Vibrio cholerae Pseudomonas aeroginosa Neisseria meningitidis Methanobacterium thermoautotrophicum Methylobacterium extorquens Pyrococcus abyssi Pyrococcus horikoshii Methanococcus jannaschii Arabidopsis thaliana Arabidopsis thaliana Arabidopsis thaliana Saccharomyces cerevisiae Schizosaccharomyces pombe Ralstonia eutropha Haemophilus influenzae Escherichia coli Synechocystis sp. Neisseria meningitidis Methanococcus jannaschii Methanobacterium thermoautotrophicum Archaeoglobus fulgidus Pyrococcus horikoshii Aeropyrum pernix Homo sapiens Caenorhabditis elegans Arabidopsis thaliana Saccharomyces cerevisiae Schizosaccharomyces pombe

Sizea (AAs)

No.b

Putative TMSc

366 372 366 360 367 322 330 302 325 314 970 890 950 459 725 499 420 552 508 426 297 314 326 406 405 516 679 587 590 574 549 527 542 424 524 494 399 426 390 390 602 759 894 538 759 620 598 656 613 658 512 276 380 380 388 756 735 617 873 898 513 461 487 449 471 432 443 397 368 430 592 545 540 894 867

spP31801 gbD90912 embCAA21344 embCAA17596 spP55471 gi2622172 gi2622261 gbU67466 dbjBAA29561 embCAB50465 spP48765 gbU70857 gbL39835 gbU57411 spP47144 spP37308 spP45268 gi1449339 embCAB73448 gi7388438 spQ58047 gi2649818 gi2623023 dbjBAA29731 embCAB50306 gbU50312 gbL20859 gbX97484 spP15710 spP38361 spP32703 dbjBAA17925 gbZ77163 dbjBAA31695 gi6714545 gi2649761 gi2621849 spQ60362 gi5458640 gi3257347 gbU21317 spQ01345 pirA31311 gbAAD16946 embCAB16384 embCAA40066 gbAAA84962 gbAAF95747 gbAAG04596 embCAB83376 gbAAB84864 gbAAA72331 embCAB50569 dbjBAA29375 gbAAB99281 embCAB80850 gbAAD03448 embCAB80872 embCAA89387 embCAB76234 spQ07252 pirI64080 gbU28379 dbjBAA18751 embCAB84272 gbU67514 gi2621879 gi2648213 dbjBAA29909 dbjBAA78964 gbU26209 spP46556 dbjBAA96091 spP27514 embCAA18293

11 11 11 11 11 10 9 10 10 10 12 10 12 10 12 11 12 12 12 11 10 9 10 11 11 14 11 13 11 10 12 11 12 11 13 13 10 12 12 11 11 12 12 11 11 12 11 11 12 13 11 7 12 12 12 12 11 12 14 12 14 14 14 12 13 12 11 12 10 12 13 11 12 13 12

Size in number of amino acyl residues (AAs). Database and accession number. Putative number of ␣-helical TMSs predicted using the TMPred program. d N and C are the N-terminal and C-terminal hydrophilic domains (regions) preceding the first predicted TMS and following the last predicted TMS, respectively, based on TMPred analyses. Region number refers to the number of the putative TMS, based on TMPred predictions. b c

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TABLE 8—Continued Residues in regiond: N

–14 1–6 1–16 1–5 1–11 1–11 1–16

1

15–33 7–25 17–35 6–24 12–32 12–28 17–33 1–18 1–10 11–29 1–2 3–20 1–8 9–25 1–3 4–22 1–2 3–22 1–40 41–57 1–22 23–42 1–9 10–29 1–9 10–26 1–39 40–58 1–5 6–24 1–17 1–11 12–32 1–2 3–20 1–2 3–21 1–5 6–23 1–4 5–21 1–3 4–20 1–20 21–41 1–126 127–143 1–22 1–7 8–24 1–2 3–21 1–16 17–38 1–10 11–33 1–19 1–8 9–27 1–8 9–29 1–2 3–22 1–2 3–21 1 2–21 1–21 1–30 31–52 1–14 15–32 1–14 15–34 1–19 20–40 1–12 13–32 1–4 5–24 1–17 1–11 12–32 1–6 7–25 1–5 6–23 1–17 1–6 7–25 1–2 3–22 1 2–22 1–2 3–25 1–83 84–105 1–83 84–105 1–19 20–37 1–17 1–32 33–54 1–33 34–51 1–12 13–30 1–9 10–29 1–3 4–22 1–28 29–47 1–6 7–27 1–14 15–35 1–19 1–12 13–34 1–18 1–13 14–31 1–25 26–45 1–46 47–65 1–384 385–403 1–405 406–426

2

3

4

5

6

7

8

9

10

11

39–58 26–46 38–57 27–45 40–56 43–62 45–63 34–52 51–69 35–54 72–93 56–77 121–142 98–121 76–98 52–72 48–64 68–90 31–47 34–57 44–63 40–60 42–62 36–60 35–59 29–45 57–77 156–174 41–62 44–64 31–50 41–62 38–58 27–45 34–51 30–47 27–45 29–49 28–47 29–48 64–81 73–89 105–121 50–67 36–56 26–46 24–44 64–82 28–51 26–49 21–40 30–52 25–44 24–44 25–45 107–126 118–139 48–66 22–40 59–78 64–80 36–55 34–57 26–47 71–97 44–64 62–82 26–44 45–65 23–40 39–58 55–75 93–112 432–448 431–453

74–94 63–82 76–92 60–79 74–90 89–108 96–115 73–97 86–102 75–93 134–151 114–134 179–199 125–141 119–138 93–112 88–107 106–123 71–87 82–100 70–90 73–92 79–96 83–102 82–101 50–72 100–122 195–215 85–105 89–110 52–71 97–114 61–81 66–84 68–84 55–71 61–78 60–82 55–74 55–74 92–115 95–114 128–146 74–96 102–125 55–74 57–74 85–105 57–78 57–76 52–74 59–78 53–70 53–70 53–71 134–154 145–166 74–93 52–71 92–110 84–100 57–74 54–74 56–74 102–122 72–91 106–130 53–74 83–106 49–68 85–105 97–121 123–143 458–478 493–514

110–126 98–115 109–128 96–116 108–128 117–136 122–142 105–126 116–132 105–123 168–187 152–172 217–238 164–180 153–173 118–138 119–137 147–165 109–129 109–132 95–115 95–115 103–121 115–134 106–128 108–128 129–147 235–253 112–132 118–139 88–107 118–134 94–110 98–121 98–115 85–102 117–137 95–112 84–106 86–106 133–151 123–142 155–174 107–129 128–152 86–110 92–108 115–135 88–111 85–109 79–95 101–120 80–98 88–108 86–103 162–181 193–212 102–126 88–108 123–144 123–143 80–103 97–116 98–114 143–159 127–147 156–174 92–112 122–140 90–113 124–140 181–197 161–179 516–534 532–555

142–161 131–147 144–163 134–152 143–162 136–156 184–204 127–143 136–156 128–145 200–219 184–206 249–271 203–220 397–415 159–177 142–164 178–200 136–156 135–157 122–140 126–144 128–147 142–159 140–158 139–159 163–180 267–285 155–171 145–163 165–183 168–187 116–140 121–140 118–137 106–122 160–178 116–137 110–133 110–128 176–194 156–179 189–213 138–155 168–192 114–133 145–164 163–183 119–138 114–135 111–133 153–175 113–133 112–133 109–129 214–233 224–243 133–149 119–140 157–175 165–185 120–140 137–157 137–156 188–208 156–175 194–213 119–138 165–184 129–149 221–242 218–238 220–241 563–581 573–591

171–188 165–182 170–188 162–180 170–192 181–200 212–228 161–177 178–195 165–184 232–249 213–233 276–298 247–267 438–459 208–227 187–205 212–233 175–194 179–195 154–174 162–180 196–212 181–200 180–199 164–182 201–223 291–309 185–206 185–205 188–207 196–214 163–182 171–189 167–185 151–170 194–212 163–181 144–168 176–198 222–243 185–205 222–243 217–237 207–227 148–166 178–200 207–231 150–168 147–171 137–156 162–215 145–169 145–169 146–166 245–264 263–282 169–189 155–173 202–221 185–203 169–189 192–210 180–199 230–250 196–216 246–266 174–193 196–217 180–196 270–291 289–307 265–288 602–621 615–639

221–240 220–236 221–240 212–228 221–241 211–232 248–267 199–219 205–226 199–216 767–789 799–820 753–769 281–300 469–491 234–251 217–234 322–343 283–301 208–227 188–204 198–218 213–231 212–230 211–227 203–221 232–251 326–350 224–244 225–244 218–236 245–263 191–212 206–224 199–217 183–200 239–260 187–205 176–198 213–229 254–274 259–279 291–311 270–288 244–264 181–203 215–236 240–257 186–205 177–200 180–201 234–254 180–200 180–200 179–197 284–303 297–318 204–223 192–216 223–241 212–228 212–231 235–258 223–241 269–287 239–257 269–287 213–233 222–243 219–237 327–345 323–339 320–336 641–664 658–676

256–276 252–271 258–276 247–268 254–273 246–264 279–299 233–256 245–263 234–252 797–815 831–850 780–796 325–345 528–550 384–400 256–274 359–377 313–331 265–283 219–238 241–261 249–269 265–284 264–283 237–254 513–529 352–371 366–385 450–469 238–260 275–296 242–263 234–253 229–246 211–230 278–298 216–233 213–229 273–291 311–329 315–335 345–362 303–320 292–314 218–239 267–287 272–289 233–254 218–236 230–248

290–310 291–314 290–310 281–302 288–309 272–294 309–329 259–275 272–292 264–281 826–845 877–896 810–832 351–371 559–581 424–444 303–320 391–407 346–364 310–330 243–260 293–310 270–290 293–311 292–310 330–346 562–578 415–433 469–488 487–507 277–295 311–328 274–292 306–328 247–266 234–251 297–318 236–259 274–291 298–320 335–358 353–374 385–407 341–358 324–348 271–290 293–312 326–344 273–292 238–255 255–272

324–343 324–342 326–343 312–335 325–342 301–321

350–366 351–367 350–366 343–360 350–366

212–231 212–231 214–231 318–339 347–369 261–277 221–241 263–281 255–274 253–270 289–307 249–266 302–320 265–284 296–313 232–252 280–301 239–260 371–392 374–392 353–371 686–702 687–704

234–252 234–252 263–279 368–390 375–394 292–308 262–284 313–336 309–329 289–309 306–328 280–298 329–345 294–311 335–353 263–279 307–327 285–305 412–432 411–429 388–406 711–730 712–732

262–280 262–280 294–314 396–415 409–432 319–339 317–337 340–362 329–350 319–335 336–354 310–334 366–382 330–350 379–396 300–323 346–365 322–339 453–471 461–484 426–442 738–756 756–778

283–299 301–322 291–308 872–891 911–929 852–870 378–396 623–641 444–465 347–366 436–457 394–411 351–369 275–293 303–325 335–354 334–353 348–366 602–620 440–461 529–547 535–557 313–336 358–376 313–330 358–376 283–301 267–285 365–385 281–301 298–320 332–350 377–395 381–400 413–432 381–399 361–380 296–315 325–343 350–368 298–317 271–289 275–295

12

13

14

368–372 367 322 323–326

906–924 943–961 881–904 926–942 668–687 476–496 369–390 484–508 436–455 399–421

C

702–724 396–415 525–548 484–500

384–405 383–404 379–401 416–435 436–453 467–489 655–677 471–488 506–527 557–576 561–581 361–379 379–399 364–387 384–402 316–334 298–317

397–420

304–328 332–350 362–382 404–424 417–437 450–469 416–435 411–433 327–346 353–371 413–429 326–346 295–316 317–337

382–400 362–382

286–304 286–304 326–345 430–453 438–457 374–396 342–364 403–423 363–380 356–374 373–395 377–399 389–406 374–394 418–438 329–351

353–373 353–373 355–374 459–478

371–394 480–496 495–516 471–492 778–796 797–818

413–429 500–520 541–563

391–409 348–366 375–392 329–347 351–369

446–466 479–499 359–378 363–381 332–353 358–375

414–431 385–401 867–886 400–419 373–394 392–415 425–445 411–430 412–430

405–424 732–753 447–467 487–507 400–417 439–460 418–437 462–480 453–471

378–397

516–534 824–842 870–889 846–862

300–302 309–314 962–970 930–955 943–950 397–427 725 497–499 416–420 549–552 501–508 422–426 294–297 311–314 326 406 405 490–516 678–679 577–587 582–590 558–574 421–549 400–527 410–542 403–424 393–524 370–494 386–399 401–426 383–390 383–390 405–602 467–759 500–894 436–538 434–759 379–620 372–598 430–656 382–613 376–658 338–512 255–276 374–380 374–380 375–388 479–756 458–735 432–617 754–873 887–898 508–513 461 481–487 446–449 431–432 439–443 366–368 430 564–592 517–545 535–540 890–894 863–867

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TABLE 9. Average size differences in various domains for the protein types analyzed Relative size (%) Protein type

Integral membrane transporters ABC permeases Receptors Membrane proteins Energizers Cytoplasmic enzymes

Bacteria Archaea

Eucarya All kingdoms Plants Animals Fungi

100

92

140

83

105

100

100 100 100 100

107 96 96 97

103

99

100

100

logues. When the three constituents of procaryotic-specific ABC-type uptake permeases were examined, the archaeal extracytoplasmic receptors proved to be 7% larger than their bacterial homologues, on average, while the membrane and cytoplasmic constituents were 3 to 4% smaller. Homologous cytoplasmic enzymes showed little or no significant difference between the three domains of life (Table 9). Within the Eucarya kingdoms, integral membrane transporters of plants proved to be significantly smaller than those of animals (21%) and fungi (17%), although no corresponding size differences were noted for homologous cytoplasmic enzymes. These observations clearly show that during evolution, integral membrane transport proteins have been subject to different pressures giving rise to size differences that are not paralleled in cytoplasmic proteins or extracytoplasmic receptors. In fact, the latter proteins exhibit significant size differences between Bacteria and Archaea that are opposite to those observed for the integral membrane proteins. These observations must be explainable at the molecular level. The molecular explanation(s) for the protein size differences documented in this report is currently elusive. Several investigators have noted that when plasmidic DNA sequences exhibiting short repetitive elements are transferred to yeast, the repeats tend to increase in number, although the reverse is true in Escherichia coli (1, 5, 6, 12). The molecular basis for this observation is not known, but if operative on chromosomal DNA over an extended period of evolutionary time, it could account for the observed average membrane protein homologue size differences. However, because the cytoplasmic proteins analyzed do not show this trend and because extracytoplasmic receptors show the opposite trend, we disfavor such an explanation. Other explanations may exist. Our domain analyses summarized in Table 8 show that the major size differences in secondary carrier proteins occur primarily in the N- and C-terminal hydrophilic extensions and specific inter-TMS loops of these integral membrane proteins, and that the locations where the major size differences occur are family specific. Sometimes the numbers of putative ␣-helical TMSs differ, but these differences may be in part artifactual and do not generally account for the size variations observed. Hydrophilic domains in transporters are known to play regulatory roles in various well-studied procaryotic and eucaryotic transport proteins (3, 14). It is possible that Eucarya have been under greater pressure to evolve regulatory domains controlling

transport than have Bacteria and that Bacteria have in turn been under greater pressure to evolve such regions than have the Archaea. If this possibility does account for the observed size differences, then plants must have been under less stringent pressure to evolve protein regulatory sequences than were animals and fungi. Moreover, cytoplasmic enzymes have not been subject to similar constraints. These observations may have predictive value for purposes of annotation. However, one can expect that multiple explanations will account for the size variations observed. It is clear that the studies reported here pose more questions than they have answered. What are the membrane structural features or mechanistic features that promote the observed size differences? Are repeated DNA sequences present in the structural genes for these proteins, and if so, do numbers of repeats contribute to or even account for the size differences observed for their protein products? What are the physiological benefits to organisms in the three domains of life to promote homologue size variation? What accounts for the size differences observed between plant transporters and those from other Eucarya? Further computational experimentation, currently in progress, will be required to provide answers to these interesting questions. ACKNOWLEDGMENTS We thank Donna Yun, Monica Mistry, Milda Simonaitis, and Yolanda Anglin for their assistance in the preparation of this manuscript. Work in the authors’ laboratory was supported by NIH grant no. 2R01 AI14176 from the National Institute of Allergy and Infectious Diseases and no. 9RO1 GM55434 from the National Institute of General Medical Sciences, as well as by the M. H. Saier, Sr. Memorial Research Fund. REFERENCES 1. Henderson, S. T., and T. D. Petes. 1992. Instability of simple sequence DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 12:2749–2757. 2. Hofmann, K., and W. Stoffel. 1993. TMPred—a database of membrane spanning protein segments. Biol. Chem. Hoppe-Seyler 347:166. 3. Hoischen, C., J. Levin, S. Pitaknarongphorn, J. Reizer, and M. H. Saier, Jr. 1996. Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system. J. Bacteriol. 178:6082–6086. 4. Kaback, H. R., and J. Wu. 1997. From membrane to molecule to the third amino acid from the left with a membrane transport protein. Q. Rev. Biophys. 30:333–364. 5. Levinson, G., and G. A. Gutman. 1987. Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4:203–221. 6. Morel, P., C. Reverdy, B. Michel, S. D. Ehrlich, and E. Cassuto. 1998. The role of SOS and flap processing in microsatellite instability in Escherichia coli. Proc. Natl. Acad. Sci. USA 95:10003–10008. 7. Persson, B. L., J. Petersson, U. Fristedt, R. Weinander, A. Berhe, and J. Pattison. 1999. Phosphate permeases of Saccharomyces cerevisiae: structure, function and regulation. Biochim. Biophys. Acta 1422:255–272. 8. Saier, M. H., Jr. 1998. Molecular phylogeny as a basis for the classification of transport proteins from bacteria, archaea and eukarya, p. 81–136. In R. K. Poole (ed.), Advances in microbial physiology. Academic Press, San Diego, Calif. 9. Saier, M. H., Jr. 1999. Genome archeology leading to the characterization and classification of transport proteins. Curr. Opin. Microbiol. 2:555–561. 10. Saier, M. H., Jr. 2000. A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol. Mol. Biol. Rev. 64:354–411. 11. Stevens, B. R., A. Fernandez, B. Hirayama, E. M. Wright, and E. S. Kempner. 1990. Intestinal brush border membrane Na⫹/glucose cotransporter functions in situ as a homotetramer. Proc. Natl. Acad. Sci. USA 87:1456– 1460. 12. Strand, M., T. A. Prolla, R. M. Liskay, and T. D. Petes. 1994. Destabilization

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of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365:274–276. 13. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882. 14. Vankeerberghen, A., W. Lin, M. Jaspers, H. Cuppens, B. Nilius, and J.-J. Cassiman. 1999. Functional characterization of the CFTR R domain using

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CFTR/MDR1 hybrid and deletion constructs. Biochemistry 38:14988–14998. 15. Verrey, F., D. L. Jack, I. T. Paulsen, M. H. Saier, Jr., and R. Pfeiffer. 1999. New glycoprotein-associated amino acid transporters. J. Membr. Biol. 172: 181–192. 16. West, I. C. 1997. Ligand conduction and the gated-pore mechanism of transmembrane transport. Biochim. Biophys. Acta 1331:213–234. 17. Zar, J. H. 1984. The Sign test (22.6), p. 386–387. In B. Kurtz (ed.), Biostatistical analysis. Prentice-Hall, Inc., Englewood Cliffs, N.J.