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.
J. Bacteriol. 2001, 183(3):1012. DOI:
10.1128/JB.183.3.1012-1021.2001.

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JOURNAL OF BACTERIOLOGY, Feb. 2001, p. 1012–1021 Vol. 183, No. 3
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.3.1012–1021.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.

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

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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 counter-
parts. 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 exhib-
ited 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.

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 transport-
ers 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 sec-
ondary 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 clas-
sification (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 fami-
lies 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 be-
tween 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-
* 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 Univer-
sity, Chonju, Korea.
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 extracel-
lular 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.
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 topolog-
ical 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 ho-
mologues 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 sec-
ondary carriers, 3 families of channel proteins, and 4 families
of primary active transporters present in the archaeal, bacte-
rial, and eucaryotic domains. The number of homologues ex-
amined 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

1012
VOL. 183, 2001 MEMBRANE PROTEINS OF BACTERIA, ARCHAEA, AND EUCARYA 1013

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

Archaea Bacteria Eucarya

Family TC no.a
No. of % Relative No. of No. of % Relative
AAsb sizec AAsb AAsb sizec

Carriers
Sugar porter (major facilitator superfamily) 2.A.1.1 399 (4) 95 422 (6) 527 (18) 124
Amino acid-polyamine-organocation 2.A.3 508 (6) 109 463 (6) 602 (5) 131
Cation diffusion facilitator 2.A.4 293 (4) 98 298 (4) 491 (8) 165
Resistance-nodulation-division 2.A.6 758 (3) 76 998 (56) 1,296 (4) 130
SecDF 2.A.6.4 713 (3) 76 935 (13) —d
Ca2⫹:cation antiporter 2.A.19 320 (4) 87 370 (7) 649 (24) 174

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Inorganic phosphate transporter 2.A.20 332 (6) 70 477 (5) 581 (10) 122
Monovalent cation:proton antiporter-1 2.A.36 440 (3) 85 516 (5) 702 (15) 136
Monovalent cation:proton antiporter-2 2.A.37 410 (5) 84 490 (13) 793 (5) 162
K⫹ transporter 2.A.38 477 (3) 99 480 (5) 758 (8) 157
Nucleobase:cation symporter-2 2.A.40 439 (3) 97 452 (9) 566 (13) 125
Formate-nitrite transporter 2.A.44 277 (2) 102 273 (7) 547 (2) 200
Divalent anion:Na⫹ symporter 2.A.47 410 (4) 79 516 (8) 681 (12) 132
Ammonium transporter 2.A.49 411 (7) 88 464 (7) 503 (12) 108
Multi-antimicrobial extrusion 2.A.66 454 (5) 99 458 (5) 636 (6) 138

Channels
Major intrinsic protein 1.A.8 246 (2) 98 251 (11) 278 (33) 111
Chloride channel 1.A.11 410 (5) 89 458 (5) 827 (17) 180
Metal ion transporter 9.A.17 330 (3) 99 332 (19) 692 (9) 210

Primary active transporters

P-type ATPase 3.A.3 724 (9) 99 732 (85) 1,096 (62) 150
Arsenite-antimonite efflux 3.A.4 388 (7) 94 411 (22) 693 (11) 169
Type II secretory pathway (SecY) 3.A.5 461 (12) 106 436 (44) 455 (26) 104
Na⫹-transporting carboxylic acid 3.B.1 385 (3) 96 399 (9) —d
decarboxylase (␤)
a
For further information on TC numbers, see reference 10 or http://www-biology.ucsd.edu/⬃msaier/transport/.
b
The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses.
c
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.

proteins were identified, all of these were also included. How-
ever, when the numbers of bacterial and/or eucaryotic homo-
logues 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 sec-
ondary 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 com-
pared 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 aver-
age, the eucaryotic proteins are 40% larger.
Size variation in integral membrane transport protein ho-
mologues in fungi, plants, and animals. Within the Eucarya
domain, animal, plant, and fungal (including yeast) homo-
logues 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 ammo-
nium 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 homo-
logues.
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
1014 CHUNG ET AL. J. BACTERIOL.

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

Animals Plants Fungi

Family TC no.
No. of % Relative No. of % Relative No. of
AAsa sizeb AAsa sizeb AAsa

Carriers
Sugar porter (major facilitator superfamily) 2.A.1.1 491 (6) 86 518 (6) 91 571 (6)
Amino acid/auxin porter 2.A.18 517 (13) 93 475 (16) 85 557 (9)
Ca2⫹:cation antiporter 2.A.19 897 (14) 147 441 (4) 72 610 (6)
Cation-chloride cotransporter 2.A.30 1,060 (14) 98 973 (1) 90 1,085 (2)
Monovalent cation:proton antiporter-1 2.A.36 755 (10) 115 488 (2) 74 657 (3)
Nucleobase:cation symporter-2 2.A.40 591 (6) 101 523 (5) 89 588 (2)

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K⫹ transporter 2.A.43 —c 506 (2) 50 1,010 (6)
Divalent anion:Na⫹ symporter 2.A.47 575 (8) 65 —c 891 (4)
Ammonium transporter 2.A.49 576 (4) 127 493 (5) 109 452 (3)

Channels
Major intrinsic protein 1.A.8 315 (15) 102 268 (22) 87 308 (2)
Chloride channel 1.A.11 858 (11) 108 778 (6) 98 796 (2)
Metal ion transporter 9.A.17 725 (3) 102 657 (4) 93 710 (2)

Primary active transporters

P-type ATPase 3.A.3 1,348 (21) 135 954 (29) 95 999 (12)
Arsenite-antimonite efflux 3.A.4 667 (9) 82 —c 809 (2)
Type II secretory pathway (SecY) 3.A.5 440 (6) 113 471 (16) 120 391 (3)
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 pro-
teins are 17% smaller.
Size variation in homologous constituents of the ABC-type
transport system in Bacteria and Archaea. The ABC superfam-
ily of uptake permeases is restricted to procaryotes, but it is
found in both Bacteria and Archaea. These systems include
three constituents: extracytoplasmic receptors, integral mem-
brane 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, respec-
tively). 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 ar-
chaeal 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 aver-
age. Overall, the archaeal cytoplasmic proteins were 3.5%
smaller than their bacterial homologues. Thus, the trend dis-
played 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 recep-
tors 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 ana-
bolic 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 ar-
chaeal 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 dif-
ferences. To test for statistically significant differences in trans-
port protein lengths between phylogenetic groups, the data
were analyzed using two-tailed Sign tests (17). In these analy-
ses, 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, nonpara-
metric 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
VOL. 183, 2001 MEMBRANE PROTEINS OF BACTERIA, ARCHAEA, AND EUCARYA 1015

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

Family TC no. Archaea Bacteria

No. of AAsa % Relative sizeb No. of AAsa % Relative sizeb

Carbohydrate uptake transporter-1 family 3.A.1.1 477 (6) 111 421 (13) 100
Carbohydrate uptake transporter-2 family 3.A.1.2 —c 343 (12) 100
Polar amino acid uptake transporter family 3.A.1.3 278 (2) 106 262 (29) 100
Hydrophobic amino acid uptake transporter family 3.A.1.4 443 (4) 118 376 (14) 100
Peptide-opine-nickel uptake transporter family 3.A.1.5 644 (11) 121 532 (25) 100
Sulfate uptake transporter family 3.A.1.6 —c 342 (6) 100
Phosphate uptake transporter family 3.A.1.7 337 (6) 101 334 (18) 100
Molybdate uptake transporter family 3.A.1.8 264 (2) 105 251 (15) 100
Phosphonate uptake transporter family 3.A.1.9 —c 300 (5) 100

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Ferric iron uptake transporter family 3.A.1.10 —c 328 (15) 100
Polyamine-opine-phosphonate uptake transporter family 3.A.1.11 417 (2) 118 352 (20) 100
Quaternary amine uptake transporter family 3.A.1.12 —c 421 (7) 100
Vitamin B12 uptake transporter family 3.A.1.13 361 (4) 129 279 (14) 100
Iron chelate uptake transporter family 3.A.1.14 347 (2) 106 327 (20) 100
Manganese-zinc-iron chelate uptake transporter family 3.A.1.15 316 (4) 103 306 (26) 100
Nitrate-nitrite-cyanate uptake transporter family 3.A.1.16 353 (4) 78 451 (10) 100
Taurine uptake transporter family 3.A.1.17 —c 333 (11) 100
Putative cobalt uptake transporter family 3.A.1.18 88 (4) 84 105 (3) 100
Thiamine uptake transporter family 3.A.1.19 350 (2) 101 346 (12) 100
Brachyspira iron transporter family 3.A.1.20 —c 346 (10) 100
a
The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses.
b
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.

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 do-
main 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 sig-
nificant. 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 TC no. Archaea Bacteria

a b a
No. of AAs % Relative size No. of AAs % Relative sizeb

Carbohydrate uptake transporter-1 family (MalF) 3.A.1.1 301 (4) 83 363 (24) 100
Carbohydrate uptake transporter-1 family (MalG) 3.A.1.1 309 (4) 96 321 (24) 100
Carbohydrate uptake transporter-2 family (RbsC) 3.A.1.2 332 (1) 97 342 (22) 100
Carbohydrate uptake transporter-2 family (RbsD) 3.A.1.2 —c 137 (7) 100
Polar amino acid uptake transporter family (HisM) 3.A.1.3 222 (2) 96 232 (50) 100
Polar amino acid uptake transporter family (HisQ) 3.A.1.3 222 (2) 95 233 (39) 100
Hydrophobic amino acid uptake transporter family (LivH) 3.A.1.4 289 (5) 91 318 (17) 100
Hydrophobic amino acid uptake transporter family (LivM) 3.A.1.4 351 (8) 87 402 (20) 100
Peptide-opine-nickel uptake transporter family (OppB) 3.A.1.5 333 (12) 103 324 (48) 100
Peptide-opine-nickel uptake transporter family (OppC) 3.A.1.5 366 (9) 116 315 (51) 100
Sulfate uptake transporter family 3.A.1.6 —c 277 (10) 100
Phosphate uptake transporter family 3.A.1.7 285 (7) 90 315 (23) 100
Molybdate uptake transporter family 3.A.1.8 248 (4) 107 232 (13) 100
Phosphonate uptake transporter family 3.A.1.9 —c 246 (5) 100
Ferric iron uptake transporter family 3.A.1.10 —c 520 (8) 100
Polyamine-opine-phosphonate uptake transporter family (PotB) 3.A.1.11 263 (3) 90 291 (25) 100
Polyamine-opine-phosphonate uptake transporter family (PotC) 3.A.1.11 262 (3) 97 271 (18) 100
Quaternary amine uptake transporter family 3.A.1.12 —c 435 (7) 100
Vitamin B12 uptake transporter family 3.A.1.13 345 (6) 101 341 (11) 100
Iron chelate uptake transporter family (FecC) 3.A.1.14 338 (6) 99 340 (18) 100
Iron chelate uptake transporter family (FecD) 3.A.1.14 344 (4) 99 346 (22) 100
Manganese-zinc-iron chelate uptake transporter family 3.A.1.15 271 (4) 93 291 (38) 100
Nitrate-nitrite-cyanate uptake transporter family 3.A.1.16 254 (5) 83 305 (16) 100
Taurine uptake transporter family 3.A.1.17 —c 328 (7) 100
Putative cobalt uptake transporter family 3.A.1.18 255 (30) 89 287 (11) 100
Thiamine uptake transporter family 3.A.1.19 406 (4) 117 348 (31) 100
Brachyspira iron transporter family 3.A.1.20 —c 424 (7) 100
a
The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses.
b
Average percent size of the archaeal homologues relative to the bacterial homologues is presented for each of the families examined.
c
No protein homologues were identified in this domain.
1016 CHUNG ET AL. J. BACTERIOL.

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

Family TC no. Archaea Bacteria

No. of AAsa % Relative sizeb No. of AAsa % Relative sizeb

Carbohydrate uptake transporter-1 family 3.A.1.1 363 (14) 99 366 (48) 100
Carbohydrate uptake transporter-2 family 3.A.1.2 496 (5) 98 504 (34) 100
Polar amino acid uptake transporter family 3.A.1.3 240 (2) 95 252 (31) 100
Hydrophobic amino acid uptake transporter family 3.A.1.4 258 (6) 97 267 (15) 100
Peptide-opine-nickel uptake transporter family 3.A.1.5 324 (9) 96 336 (25) 100
Sulfate uptake transporter family 3.A.1.6 329 (1) 92 357 (8) 100
Phosphate uptake transporter family 3.A.1.7 286 (10) 92 312 (21) 100
Molybdate uptake transporter family 3.A.1.8 344 (1) 99 349 (8) 100

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Phosphonate uptake transporter family 3.A.1.9 —c 287 (4) 100
Ferric iron uptake transporter family 3.A.1.10 329 (1) 93 353 (9) 100
Polyamine-opine-phosphonate uptake transporter family 3.A.1.11 343 (5) 91 376 (13) 100
Quaternary amine uptake transporter family 3.A.1.12 369 (3) 93 396 (12) 100
Vitamin B12 uptake transporter family 3.A.1.13 252 (3) 96 263 (10) 100
Iron chelate uptake transporter family 3.A.1.14 272 (2) 101 269 (24) 100
Manganese-zinc-iron chelate uptake transporter family 3.A.1.15 271 (3) 103 262 (21) 100
Nitrate-nitrite-cyanate uptake transporter family 3.A.1.16 250 (3) 91 274 (18) 100
Taurine uptake transporter family 3.A.1.17 —c 332 (11) 100
Putative cobalt uptake transporter family 3.A.1.18 284 (9) 103 276 (11) 100
Thiamine uptake transporter family 3.A.1.19 —c 225 (3) 100
Brachyspira iron transporter family 3.A.1.20 —c 371 (3) 100
a
The average number of amino acyl residues (AAs) per protein homologue is reported, with the number of proteins examined appearing in parentheses.
b
Average percent size of the archaeal homologue relative to the bacterial homologue is presented for each of the families examined.
c
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 Eu-
carya. Tests were generally less significant in pairwise compar-
isons of Eucarya kingdoms. Plants have shorter transport pro-
teins than either animals or fungi, but the difference in protein
length between animals and fungi is not significant. Corre-
sponding 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 trans-
porters responsible for size differences between Bacteria, Ar-
chaea, 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 se-
quence-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 pre-
dicted 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
Eucarya
Enzyme EC no. Archaea Bacteria
All kingdoms Animals Plants Fungi

Enolase 4.2.1.11 425 (6) 430 (23) 438 (30) 434 (13) 444 (8) 438 (9)
Phosphoglycerate kinase 2.7.2.3 409 (10) 398 (21) 414 (27) 416 (11) 401 (4) 417 (12)
Glyceraldehyde 3-phosphate dehydrogenase 1.2.1.12 338 (12) 335 (24) 336 (57) 334 (23) 339 (13) 335 (21)
Triose-phosphate isomerase 5.3.1.1 224 (10) 253 (25) 251 (24) 249 (11) 254 (8) 249 (5)
Phosphoglucose isomerase 5.3.1.9 401 (1) 517 (22) 560 (18) 557 (7) 566 (8) 554 (3)
Pyruvate kinase 2.1.7.40 465 (4) 500 (22) 522 (22) 529 (9) 509 (6) 524 (7)
Inosine-5⬘-monophosphate dehydrogenase 1.1.1.250 481 (7) 500 (16) 517 (11) 520 (4) 502 (3) 525 (4)
Inosine-5⬘-monophosphate-aspartate ligase 6.3.4.4 340 (6) 426 (19) 447 (6) 454 (4) —a 434 (2)
Glutamine synthetase 6.3.1.2 451 (11) 466 (26) 369 (33) 376 (13) 365 (15) 360 (5)
Aspartate aminotransferase 2.6.1.1 391 (6) 394 (17) 412 (15) 412 (8) 409 (5) 421 (2)
Elongation factor-2/elongation factor-G No EC no. 731 (16) 698 (25) 849 (12) 855 (6) 845 (3) 842 (3)
a
No sequenced homologues of this enzyme were found in plants.
VOL. 183, 2001 MEMBRANE PROTEINS OF BACTERIA, ARCHAEA, AND EUCARYA
TABLE 7. Results of statistical analyses of observed homologue size differences
Homologue Groups compared
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)
ABC receptor Archaea (A) vs Bacteria (B)
ABC membrane protein Archaea (A) vs Bacteria (B)
ABC cytoplasmic energizer Archaea (A) vs Bacteria (B)
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 exper-
imental 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 pro-
teins (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, ac-
counting 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 pro-
teins 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 pro-
gram 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 phos-
phate 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 vari-
ations observed between the procaryotic and eucaryotic pro-
teins 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 resi-
dues), 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
1017
No. with
Sign test P value Correlation
A ⬎ B:A ⬍ B
3:19 0.002 A⬍B
0:20 ⬍0.001 A⬍B
19:1 ⬍0.001 A⬎B
2:10 0.039 A⬍B
9:5 0.42 None
11:2 0.022 A⬎B
11:2 0.022 A⬎B
5:15 0.041 A⬍B
3:13 0.013 A⬍B
Downloaded from http://jb.asm.org/ on October 26, 2014 by UNIV OF VICTORIA
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 do-
mains, but their C-terminal hydrophilic domains differ substan-
tially 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⫹ sym-
porter 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 homo-
logues 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 N-
terminal hydrophilic domains, others show differences in the
C-terminal hydrophilic domains, and still others show differ-
ences in specific inter-TMS loop regions. Most families exhibit
size differences between homologues that represent a combi-
nation 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 trans-
port proteins of bacteria are 8% larger than their archaeal
homologues and 40% smaller than their eucaryotic homo-
1018 CHUNG ET AL. J. BACTERIOL.

TABLE 8. Localization of regions of size difference between homologues of five families of secondary carriers

Homologue Sizea Putative

family (TC) Group Name Organism No.b
(AAs) TMSc

Ca2⫹:cation antiporter Bacteria ChaA Eco Escherichia coli 366 spP31801 11

(2.A.19) CaHA Syn Synechocystis sp. 372 gbD90912 11
Orf21 Ype Yersinia pestis 366 embCAA21344 11
ChaA Mtu Mycobacterium tuberculosis 360 embCAA17596 11
Y4HA Rhi Rhizobium sp. 367 spP55471 11
Archaea Orf1 Mth Methanobacterium thermoautotrophicum 322 gi2622172 10
Orf2 Mth Methanobacterium thermoautotrophicum 330 gi2622261 9
Orf Mja Methanococcus jannaschii 302 gbU67466 10
Orf Pho Pyrococcus horikoshii 325 dbjBAA29561 10
Orf Pab Pyrococcus abyssi 314 embCAB50465 10
Eucarya CaSA2 Bta Bos taurus 970 spP48765 12
Orf1 Cel Caenorhabditis elegans 890 gbU70857 10

Downloaded from http://jb.asm.org/ on October 26, 2014 by UNIV OF VICTORIA

CaSA Dme Drosophila melanogaster 950 gbL39835 12
CAX1 Ath Arabidopsis thaliana 459 gbU57411 10
Orf3 Sce Saccharomyces cerevisiae 725 spP47144 12
Inorganic phosphate Bacteria PitA Eco Escherichia coli 499 spP37308 11
transporter (2.A.20) YG04 Hin Haemophilus influenzae 420 spP45268 12
PitB Mtu Mycobacterium tuberculosis 552 gi1449339 12
Orf Cje Campylobacter jejuni 508 embCAB73448 12
Orf Cpn Chlamydophila pneumoniae 426 gi7388438 11
Archaea Y630 Mja Methanococcus jannaschii 297 spQ58047 10
Orf Afu Archaeoglobus fulgidus 314 gi2649818 9
Orf Mth Methanobacterium thermoautotrophicum 326 gi2623023 10
Orf Pho Pyrococcus horikoshii 406 dbjBAA29731 11
Orf Pab Pyrococcus abyssi 405 embCAB50306 11
Eucarya Orf Cel Caenorhabditis elegans 516 gbU50312 14
Glvr Hsa Homo sapiens 679 gbL20859 11
Pit Ath Arabidopsis thaliana 587 gbX97484 13
Pho4 Ncr Neurospora crassa 590 spP15710 11
Npa Sce Saccharomyces cerevisiae 574 spP38361 10
Monovalent cation:proton Bacteria YjcE Eco Escherichia coli 549 spP32703 12
antiporter-1 (2.A.36) Orf Syn Synechocystis sp. 527 dbjBAA17925 11
Orf Mtu Mycobacterium tuberculosis 542 gbZ77163 12
NhaP Pae Pseudomonas aeroginosa 424 dbjBAA31695 11
Orf Bsu Bacillus subtilis 524 gi6714545 13
Archaea Nhe2 Afu Archaeoglobus fulgidus 494 gi2649761 13
Orf Mth Methanobacterium thermoautotrophicum 399 gi2621849 10
Y057 Mja Methanococcus jannaschii 426 spQ60362 12
Orf Pab Pyrococcus abyssi 390 gi5458640 12
Orf Pho Pyrococcus horikoshii 390 gi3257347 11
Eucarya Orf Cel Caenorhabditis elegans 602 gbU21317 11
NahB Cmy Oncorhynchus mykiss 759 spQ01345 12
Nhel Hsa Homo sapiens 894 pirA31311 12
Nhx1 Ath Arabidopsis thaliana 538 gbAAD16946 11
Orf Spo Schizosaccharomyces pombe 759 embCAB16384 11
Monovalent cation:proton Bacteria KefC Eco Escherichia coli 620 embCAA40066 12
antiporter-2 (2.A.37) KefC Mxa Mixococcus xanthus 598 gbAAA84962 11
KefB Vch Vibrio cholerae 656 gbAAF95747 11
Orf Pae Pseudomonas aeroginosa 613 gbAAG04596 12
Orf Nme Neisseria meningitidis 658 embCAB83376 13
Archaea Orf Mth Methanobacterium thermoautotrophicum 512 gbAAB84864 11
PhaC Mex Methylobacterium extorquens 276 gbAAA72331 7
Orf Pab Pyrococcus abyssi 380 embCAB50569 12
Orf Pho Pyrococcus horikoshii 380 dbjBAA29375 12
Orf Mja Methanococcus jannaschii 388 gbAAB99281 12
Eucarya Orf1 Ath Arabidopsis thaliana 756 embCAB80850 12
Orf2 Ath Arabidopsis thaliana 735 gbAAD03448 11
Orf3 Ath Arabidopsis thaliana 617 embCAB80872 12
Orf Sce Saccharomyces cerevisiae 873 embCAA89387 14
Orf Spo Schizosaccharomyces pombe 898 embCAB76234 12
Divalent anion:Na⫹ Bacteria Orf Reu Ralstonia eutropha 513 spQ07252 14
symporter (2.A.47) Orf Hin Haemophilus influenzae 461 pirI64080 14
YbdS Eco Escherichia coli 487 gbU28379 14
Orf Syn Synechocystis sp. 449 dbjBAA18751 12
Orf Nme Neisseria meningitidis 471 embCAB84272 13
Archaea Orf Mja Methanococcus jannaschii 432 gbU67514 12
Orf Mth Methanobacterium thermoautotrophicum 443 gi2621879 11
Orf Afu Archaeoglobus fulgidus 397 gi2648213 12
Orf Pho Pyrococcus horikoshii 368 dbjBAA29909 10
Orf Ape Aeropyrum pernix 430 dbjBAA78964 12
Eucarya NadC Hsa Homo sapiens 592 gbU26209 13
Orf Cel Caenorhabditis elegans 545 spP46556 11
Orf Ath Arabidopsis thaliana 540 dbjBAA96091 12
Orf Sce Saccharomyces cerevisiae 894 spP27514 13
Orf Spo Schizosaccharomyces pombe 867 embCAA18293 12
a
Size in number of amino acyl residues (AAs).
b
Database and accession number.
c
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.
VOL. 183, 2001 MEMBRANE PROTEINS OF BACTERIA, ARCHAEA, AND EUCARYA 1019

TABLE 8—Continued
Residues in regiond:

N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 C

–14 15–33 39–58 74–94 110–126 142–161 171–188 221–240 256–276 290–310 324–343 350–366
1–6 7–25 26–46 63–82 98–115 131–147 165–182 220–236 252–271 291–314 324–342 351–367 368–372
1–16 17–35 38–57 76–92 109–128 144–163 170–188 221–240 258–276 290–310 326–343 350–366
1–5 6–24 27–45 60–79 96–116 134–152 162–180 212–228 247–268 281–302 312–335 343–360
1–11 12–32 40–56 74–90 108–128 143–162 170–192 221–241 254–273 288–309 325–342 350–366 367
1–11 12–28 43–62 89–108 117–136 136–156 181–200 211–232 246–264 272–294 301–321 322
1–16 17–33 45–63 96–115 122–142 184–204 212–228 248–267 279–299 309–329
1–18 34–52 73–97 105–126 127–143 161–177 199–219 233–256 259–275 283–299 300–302
1–10 11–29 51–69 86–102 116–132 136–156 178–195 205–226 245–263 272–292 301–322 323–326
1–2 3–20 35–54 75–93 105–123 128–145 165–184 199–216 234–252 264–281 291–308 309–314
1–8 9–25 72–93 134–151 168–187 200–219 232–249 767–789 797–815 826–845 872–891 906–924 943–961 962–970
1–3 4–22 56–77 114–134 152–172 184–206 213–233 799–820 831–850 877–896 911–929 930–955

Downloaded from http://jb.asm.org/ on October 26, 2014 by UNIV OF VICTORIA

1–2 3–22 121–142 179–199 217–238 249–271 276–298 753–769 780–796 810–832 852–870 881–904 926–942 943–950
1–40 41–57 98–121 125–141 164–180 203–220 247–267 281–300 325–345 351–371 378–396 397–427
1–22 23–42 76–98 119–138 153–173 397–415 438–459 469–491 528–550 559–581 623–641 668–687 702–724 725
1–9 10–29 52–72 93–112 118–138 159–177 208–227 234–251 384–400 424–444 444–465 476–496 497–499
1–9 10–26 48–64 88–107 119–137 142–164 187–205 217–234 256–274 303–320 347–366 369–390 396–415 416–420
1–39 40–58 68–90 106–123 147–165 178–200 212–233 322–343 359–377 391–407 436–457 484–508 525–548 549–552
1–5 6–24 31–47 71–87 109–129 136–156 175–194 283–301 313–331 346–364 394–411 436–455 484–500 501–508
1–17 34–57 82–100 109–132 135–157 179–195 208–227 265–283 310–330 351–369 399–421 422–426
1–11 12–32 44–63 70–90 95–115 122–140 154–174 188–204 219–238 243–260 275–293 294–297
1–2 3–20 40–60 73–92 95–115 126–144 162–180 198–218 241–261 293–310 311–314
1–2 3–21 42–62 79–96 103–121 128–147 196–212 213–231 249–269 270–290 303–325 326
1–5 6–23 36–60 83–102 115–134 142–159 181–200 212–230 265–284 293–311 335–354 384–405 406
1–4 5–21 35–59 82–101 106–128 140–158 180–199 211–227 264–283 292–310 334–353 383–404 405
1–3 4–20 29–45 50–72 108–128 139–159 164–182 203–221 237–254 330–346 348–366 379–401 416–435 436–453 467–489 490–516
1–20 21–41 57–77 100–122 129–147 163–180 201–223 232–251 513–529 562–578 602–620 655–677 678–679
1–126 127–143 156–174 195–215 235–253 267–285 291–309 326–350 352–371 415–433 440–461 471–488 506–527 557–576 577–587
1–22 41–62 85–105 112–132 155–171 185–206 224–244 366–385 469–488 529–547 561–581 582–590
1–7 8–24 44–64 89–110 118–139 145–163 185–205 225–244 450–469 487–507 535–557 558–574
1–2 3–21 31–50 52–71 88–107 165–183 188–207 218–236 238–260 277–295 313–336 361–379 397–420 421–549
1–16 17–38 41–62 97–114 118–134 168–187 196–214 245–263 275–296 311–328 358–376 379–399 400–527
1–10 11–33 38–58 61–81 94–110 116–140 163–182 191–212 242–263 274–292 313–330 364–387 391–409 410–542
1–19 27–45 66–84 98–121 121–140 171–189 206–224 234–253 306–328 358–376 384–402 403–424
1–8 9–27 34–51 68–84 98–115 118–137 167–185 199–217 229–246 247–266 283–301 316–334 348–366 375–392 393–524
1–8 9–29 30–47 55–71 85–102 106–122 151–170 183–200 211–230 234–251 267–285 298–317 329–347 351–369 370–494
1–2 3–22 27–45 61–78 117–137 160–178 194–212 239–260 278–298 297–318 365–385 386–399
1–2 3–21 29–49 60–82 95–112 116–137 163–181 187–205 216–233 236–259 281–301 304–328 382–400 401–426
1 2–21 28–47 55–74 84–106 110–133 144–168 176–198 213–229 274–291 298–320 332–350 362–382 383–390
1–21 29–48 55–74 86–106 110–128 176–198 213–229 273–291 298–320 332–350 362–382 383–390
1–30 31–52 64–81 92–115 133–151 176–194 222–243 254–274 311–329 335–358 377–395 404–424 405–602
1–14 15–32 73–89 95–114 123–142 156–179 185–205 259–279 315–335 353–374 381–400 417–437 446–466 467–759
1–14 15–34 105–121 128–146 155–174 189–213 222–243 291–311 345–362 385–407 413–432 450–469 479–499 500–894
1–19 20–40 50–67 74–96 107–129 138–155 217–237 270–288 303–320 341–358 381–399 416–435 436–538
1–12 13–32 36–56 102–125 128–152 168–192 207–227 244–264 292–314 324–348 361–380 411–433 434–759
1–4 5–24 26–46 55–74 86–110 114–133 148–166 181–203 218–239 271–290 296–315 327–346 359–378 379–620
1–17 24–44 57–74 92–108 145–164 178–200 215–236 267–287 293–312 325–343 353–371 372–598
1–11 12–32 64–82 85–105 115–135 163–183 207–231 240–257 272–289 326–344 350–368 413–429 430–656
1–6 7–25 28–51 57–78 88–111 119–138 150–168 186–205 233–254 273–292 298–317 326–346 363–381 382–613
1–5 6–23 26–49 57–76 85–109 114–135 147–171 177–200 218–236 238–255 271–289 295–316 332–353 358–375 376–658
1–17 21–40 52–74 79–95 111–133 137–156 180–201 230–248 255–272 275–295 317–337 338–512
1–6 7–25 30–52 59–78 101–120 153–175 162–215 234–254 255–276
1–2 3–22 25–44 53–70 80–98 113–133 145–169 180–200 212–231 234–252 262–280 286–304 353–373 374–380
1 2–22 24–44 53–70 88–108 112–133 145–169 180–200 212–231 234–252 262–280 286–304 353–373 374–380
1–2 3–25 25–45 53–71 86–103 109–129 146–166 179–197 214–231 263–279 294–314 326–345 355–374 375–388
1–83 84–105 107–126 134–154 162–181 214–233 245–264 284–303 318–339 368–390 396–415 430–453 459–478 479–756
1–83 84–105 118–139 145–166 193–212 224–243 263–282 297–318 347–369 375–394 409–432 438–457 458–735
1–19 20–37 48–66 74–93 102–126 133–149 169–189 204–223 261–277 292–308 319–339 374–396 414–431 432–617
1–17 22–40 52–71 88–108 119–140 155–173 192–216 221–241 262–284 317–337 342–364 385–401 405–424 732–753 754–873
1–32 33–54 59–78 92–110 123–144 157–175 202–221 223–241 263–281 313–336 340–362 403–423 867–886 887–898
1–33 34–51 64–80 84–100 123–143 165–185 185–203 212–228 255–274 309–329 329–350 363–380 400–419 447–467 487–507 508–513
1–12 13–30 36–55 57–74 80–103 120–140 169–189 212–231 253–270 289–309 319–335 356–374 373–394 400–417 439–460 461
1–9 10–29 34–57 54–74 97–116 137–157 192–210 235–258 289–307 306–328 336–354 373–395 392–415 418–437 462–480 481–487
1–3 4–22 26–47 56–74 98–114 137–156 180–199 223–241 249–266 280–298 310–334 377–399 425–445 446–449
1–28 29–47 71–97 102–122 143–159 188–208 230–250 269–287 302–320 329–345 366–382 389–406 411–430 453–471
1–6 7–27 44–64 72–91 127–147 156–175 196–216 239–257 265–284 294–311 330–350 374–394 412–430 431–432
1–14 15–35 62–82 106–130 156–174 194–213 246–266 269–287 296–313 335–353 379–396 418–438 439–443
1–19 26–44 53–74 92–112 119–138 174–193 213–233 232–252 263–279 300–323 329–351 378–397
1–12 13–34 45–65 83–106 122–140 165–184 196–217 222–243 280–301 307–327 346–365 366–368
1–18 23–40 49–68 90–113 129–149 180–196 219–237 239–260 285–305 322–339 371–394 413–429 430
1–13 14–31 39–58 85–105 124–140 221–242 270–291 327–345 371–392 412–432 453–471 480–496 500–520 541–563 564–592
1–25 26–45 55–75 97–121 181–197 218–238 289–307 323–339 374–392 411–429 461–484 495–516 517–545
1–46 47–65 93–112 123–143 161–179 220–241 265–288 320–336 353–371 388–406 426–442 471–492 516–534 535–540
1–384 385–403 432–448 458–478 516–534 563–581 602–621 641–664 686–702 711–730 738–756 778–796 824–842 870–889 890–894
1–405 406–426 431–453 493–514 532–555 573–591 615–639 658–676 687–704 712–732 756–778 797–818 846–862 863–867
1020 CHUNG ET AL.
TABLE 9. Average size differences in various domains
for the protein types analyzed
Relative size (%)
Protein type All Eucarya
Bacteria Archaea
kingdoms Plants Animals Fungi
Integral membrane 100 92 140 83 105
transporters
ABC permeases
Receptors 100 107
Membrane proteins 100 96
Energizers 100 96
Cytoplasmic enzymes 100 97 103 99 100
logues. When the three constituents of procaryotic-specific
ABC-type uptake permeases were examined, the archaeal ex-
tracytoplasmic 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 Euca-
rya 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 observa-
tions 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 cyto-
plasmic proteins or extracytoplasmic receptors. In fact, the
latter proteins exhibit significant size differences between Bac-
teria and Archaea that are opposite to those observed for the
integral membrane proteins. These observations must be ex-
plainable at the molecular level.
The molecular explanation(s) for the protein size differences
documented in this report is currently elusive. Several investi-
gators have noted that when plasmidic DNA sequences exhib-
iting 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 homo-
logue size differences. However, because the cytoplasmic pro-
teins analyzed do not show this trend and because extracyto-
plasmic receptors show the opposite trend, we disfavor such an
explanation.
Other explanations may exist. Our domain analyses summa-
rized in Table 8 show that the major size differences in sec-
ondary carrier proteins occur primarily in the N- and C-termi-
nal 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 gener-
ally account for the size variations observed. Hydrophilic do-
mains in transporters are known to play regulatory roles in
various well-studied procaryotic and eucaryotic transport pro-
teins (3, 14). It is possible that Eucarya have been under
greater pressure to evolve regulatory domains controlling
J. BACTERIOL.
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 strin-
gent pressure to evolve protein regulatory sequences than were
animals and fungi. Moreover, cytoplasmic enzymes have not
100 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
100 than they have answered. What are the membrane structural
Downloaded from http://jb.asm.org/ on October 26, 2014 by UNIV OF VICTORIA
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 physiolog-
ical benefits to organisms in the three domains of life to pro-
mote 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 manu-
script.
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 Gen-
eral Medical Sciences, as well as by the M. H. Saier, Sr. Memorial
Research Fund.
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