Comparative

molecular

biological

analysis

of

membrane

transport

genes

in

organisms

Comparative

analyses

of

membrane

transport

genes

revealed

many

differences

in

the

features

of

transport

homeostasis

in

eight

diverse

organisms

ranging

from

bacteria

to

animals

and

plants

In

bacteria

membrane-transport

systems

depend

mainly

on

single

genes

encoding

proteins

involved

in

an

ATP-dependent

pump

and

secondary

transport

proteins

that

use

H

as

a

co-transport

molecule

Animals

are

especially

divergent

in

their

channel

genes

and

plants

have

larger

numbers

of

P-type

ATPase

and

secondary

active

transporters

than

do

other

organisms

The

secondary

transporter

genes

have

diverged

evolutionarily

in

both

animals

and

plants

for

different

co-transporter

molecules

Animals

use

ions

for

the

formation

of

concentration

gradients

across

plasma

membranes

dependent

on

secondary

active

transporters

and

on

membrane

voltages

that

in

turn

are

dependent

on

ion

transport

regulation

systems

Plants

use

H

ions

pooled

in

vacuoles

and

the

apoplast

to

transport

various

substances

these

proton

gradients

are

also

dependent

on

secondary

active

transporters

We

also

compared

the

numbers

of

membrane

transporter

genes

in

Arabidopsis

and

rice

Although

many

transporter

genes

are

similar

in

these

plants

Arabidopsis

has

a

more

diverse

array

of

genes

for

multi-efflux

transport

and

for

response

to

stress

signals

and

rice

has

more

secondary

transporter

genes

for

carbohydrate

and

nutrient

transport

Introduction

Cells

maintain

their

biological

activities

by

importing

and

exporting

various

substances

Provision

of

energy

and

nutrients

and

efflux

of

salts

biochemicals

and

ions

are

necessary

to

maintaining

biological

activity

in

prokaryotic

and

eukaryotic

cells

Environmental

situations

within

cells

differ

among

organisms

unicellular

organisms

can

not

control

the

ion

concentrations

outside

the

cell

but

multicellular

eukaryotes

-LRB-

especially

animals

-RRB-

can

precisely

regulate

the

ion

concentrations

of

their

cellular

environments

within

micromolar

ranges

Therefore

we

can

expect

organisms

to

differ

in

gene

number

structure

and

function

according

to

their

biological

abilities

and

environmental

situations

Recent

sequence

analyses

of

entire

genomes

have

made

it

possible

to

confirm

the

existence

of

homologous

genes

by

computer

data

analysis

It

is

also

possible

to

reveal

the

overall

patterns

of

gene

networks

In

plants

complete

genomic

sequences

are

available

for

Arabidopsis

and

rice

but

gene

annotation

programs

are

not

yet

sufficiently

accurate

to

determine

the

function

of

all

genes

Instead

full-length

cDNA

data

are

useful

for

precise

analysis

of

genes

Because

transport

activities

are

required

at

distinct

levels

in

most

tissues

we

expected

that

the

transcripts

of

most

transmembrane

transporters

would

be

represented

in

full-length

cDNA

libraries

from

plants

at

various

developmental

stages

various

plant

tissues

and

plants

exposed

to

various

treatments

We

searched

for

orthologs

of

known

membrane

transport

genes

by

using

the

full-length

rice

cDNA

sequences

-LRB-

Rice

Full-Length

cDNA

Consortium

Satoh

et

al.

-RRB-

and

genomic

sequence

data

from

Arabidopsis

-LRB-

Arabidopsis

Genome

Initiative

-RRB-

and

japonica

rice

-LRB-

Goff

et

al.

International

Rice

Genome

Sequencing

Project

-RRB-

and

global

functional

gene

annotation

in

Arabidopsis

and

rice

-LRB-

Munich

Information

Center

for

Protein

Sequences

-LRB-

MIPS

-RRB-

data

service

-LRB-

-RRB-

-LRB-

Schoof

et

al.

Karlowski

et

al.

-RRB-

-RRB-

-LRB-

RAP-DB

Rice

Annotation

Project

Data

Base

-LRB-

Rice

Annotation

Project

-RRB-

-RRB-

the

TIGR

Rice

Genome

Annotation

-LRB-

Ouyang

et

al.

-RRB-

Transmembrane

proteins

have

a

hydrophobic

structure

a

pore-forming

sequence

and

molecule-binding

sites

Because

of

these

specific

structural

features

the

identification

of

membrane

transport

orthologs

is

clear

from

computer

calculations

Previous

reports

have

characterized

individual

transporter

protein

families

but

have

not

extended

to

whole

transport

systems

in

general

-LRB-

Eng

et

al.

Pao

et

al.

Mäser

et

al.

Sánchez-Fernández

et

al.

-RRB-

In

a

more

general

analysis

of

various

organisms

the

features

of

prokaryotes

were

contrasted

with

those

of

eukaryotes

-LRB-

Ren

and

Paulsen

-RRB-

However

differences

among

eukaryotes

especially

animals

and

plants

were

not

a

focus

of

that

report

We

also

searched

for

orthologs

of

membrane

transport

gene

in

various

organisms

database

-LRB-

The

Human

Gene

Nomenclature

Database

Search

Engine

-LRB-

-RRB-

-LRB-

Wain

et

al.

-RRB-

Genomic

comparison

of

membrane

transport

systems

-LRB-

TransportDB

-RRB-

-LRB-

Ren

et

al.

-RRB-

-RRB-

the

functional

genomics

of

plant

transporters

-LRB-

PlantsT

-LRB-

Tchieu

et

al.

-RRB-

and

ARAMEMNON

-LRB-

-RRB-

-LRB-

Schwacke

et

al.

-RRB-

-RRB-

Here

we

compare

total

membrane

transport

systems

from

diverse

organisms

and

conclude

that

membrane

transport

genes

exemplify

evolutionary

diversity

of

homeostatic

systems

Evolutionary

changes

in

gene

families

indicate

the

dynamics

of

alterations

in

biological

systems

and

gene

networks

Therefore

analysis

of

large

categories

of

gene

families

may

reveal

many

basic

concepts

of

biological

systems

General

comparisons

of

membrane

transport

genes

Membrane

transport

protein

carries

various

materials

for

homeostasis

There

are

many

clear

features

of

domains

-LRB-

ex

Transmembrane

pore-forming

ATP

binding

molecular

capture

-RRB-

and

functional

features

in

the

transport

proteins

According

to

the

structure

and

its

functional

systems

the

membrane

transport

proteins

have

divided

to

three

categories-pump

channel

and

secondary

transporter

The

pump

system

is

the

slowest

-LRB-

molecules/s

-RRB-

but

environmental-independent

system

which

consume

energy

-LRB-

mainly

ATP

-RRB-

for

transport

The

channel

is

the

most

rapid

-LRB-

molecules/s

-RRB-

and

non-energy

consuming

systems

but

its

need

concentration

gradients

previously

-LRB-

transport

directions

are

only

according

to

the

gradients

-RRB-

The

secondary

transport

system

adapts

co-transport

molecules

movement

energy

to

carry

molecules

Therefore

it

needs

co-transport

molecules

and

the

transport

directions

have

depended

on

environmental

conditions

and

the

speeds

-LRB-

molecules/s

-RRB-

are

the

middle

of

pump

and

channel

We

summarized

all

three

categories

-LRB-

pump

channel

and

secondary

transporter

-RRB-

of

genes

and

compared

the

total

numbers

of

membrane

transport

genes

in

Escherichia

coli

Arabidopsis

thaliana

Oryza

sativa

Caenorhabditis

elegans

Drosophila

melanogaster

Homo

sapiens

Neurospora

crassa

and

Saccharomyces

cerevisiae

The

genome

sizes

among

these

organisms

were

diverse

-LRB-

megabases

-RRB-

and

the

numbers

of

transmembrane

genes

ranged

from

to

in

E.

coli

fungi

and

yeast

to

about

in

Arabidopsis

and

rice

-LRB-

Table

-RRB-

This

indicates

that

a

minimum

number

of

about

gene

species

may

be

required

to

retain

cell

homeostasis

The

greater

numbers

of

transmembrane

transport

genes

in

Arabidopsis

and

rice

may

indicate

additional

redundancy

as

well

as

the

modification

of

genes

for

new

roles

-LRB-

e.g.

addition

of

new

substances

adaptation

of

systems

for

regulating

transport

divergence

of

stage

and

tissue-specific

material

transport

-RRB-

specialization

for

the

various

tissues

and

cells

of

multicellular

organisms

and

increased

complexity

of

cells

which

in

eukaryotes

have

many

additional

organelles

The

greater

relative

increase

-LRB-

plants

versus

bacterium

fungus

and

yeast

-RRB-

in

the

numbers

of

membrane

transport

genes

was

less

than

has

been

reported

for

other

gene

categories

such

as

transcription

factor

genes

and

metabolic

enzyme

genes

in

higher

eukaryotes

-LRB-

Wray

et

al.

-RRB-

This

may

indicate

that

adaptations

in

membrane

transport

are

critical

for

the

survival

of

organisms

during

evolution

The

total

numbers

of

membrane

transport

genes

in

higher

plants

-LRB-

Arabidopsis

about

rice

Table

-RRB-

are

times

those

in

animals

-LRB-

fly

nematode

human

Table

-RRB-

These

differences

in

numbers

of

transporter

genes

may

be

related

to

differences

in

the

need

for

efflux

and

influx

systems

in

restricted

habitation

environments

Because

of

their

immobility

and

the

simplicity

of

their

uptake

systems

plant

cells

have

more

opportunity

to

absorb

inappropriate

substances

and

greater

amounts

of

substrates

and

to

synthesize

larger

amounts

of

secondary

products

than

do

animals

In

accordance

with

their

structures

and

mechanisms

of

action

transport

proteins

are

classified

into

three

classes

pump

channel

and

secondary

transporter

-LRB-

transporter

-RRB-

The

composition

ratios

of

these

classes

of

protein

were

also

compared

-LRB-

Table

Fig.

-RRB-

The

numbers

of

pump

genes

in

animals

-LRB-

-RRB-

were

almost

the

same

as

in

bacteria

-LRB-

-RRB-

The

numbers

of

secondary

transporter

-LRB-

animals

bacteria

-RRB-

and

channel

-LRB-

animals

bacteria

-RRB-

genes

were

increased

in

animals

In

particular

vertebrates

-LRB-

humans

-RRB-

had

more

-LRB-

-RRB-

channel

gene

species

than

plants

-LRB-

-RRB-

We

considered

that

this

gene

diversity

in

the

development

of

channel

systems

was

caused

by

the

acquisition

of

a

nervous

system

The

electrical

transmission

systems

in

nervous

systems

supplying

organs

-LRB-

e.g.

muscles

kidneys

-RRB-

need

precisely

controlled

ion

concentrations

and

the

ability

to

make

immediate

changes

in

gradients

The

development

of

active

transport

systems

in

animals

allowed

the

regulation

of

rapid

movements

of

the

body

and

organs

Therefore

animals

presumably

acquired

genes

for

the

fastest

transport-system

channels

Plants

also

had

more

channel

gene

species

than

bacteria

although

fewer

than

animals

Because

plants

do

not

transmit

signals

for

quick

movement

of

their

organs

they

do

not

need

to

regulate

membrane

voltages

as

precisely

as

animals

Additionally

signal-transmitting

systems

with

ligand

molecules

-LRB-

e.g.

neurotransmitters

-RRB-

are

not

specific

unlike

in

animals

Therefore

the

numbers

of

voltage-gated

ion

channels

-LRB-

VICs

-RRB-

and

ligand-dependent

channels

were

smaller

in

plants

than

in

higher

animals

-LRB-

Table

-RRB-

On

the

other

hand

higher

plants

had

increased

numbers

of

genes

for

pumps

-LRB-

-RRB-

and

secondary

active

transporters

-LRB-

-RRB-

Plant

cells

have

chloroplasts

which

synthesize

carbohydrates

for

many

biological

activities

including

protein

synthesis

and

functioning

of

the

ATP-dependent

pumps

Plants

presumably

use

ATP-consuming

systems

more

easily

than

animals

and

the

pumps

transport

the

molecules

that

act

as

the

driving

forces

of

the

secondary

active

transporters

Additionally

plant-specific

organelles

and

vacuoles

provide

pools

of

ions

and

catabolite

molecules

Co-transport

molecules

for

secondary

transport

also

are

safely

and

stably

stored

in

the

vacuoles

Therefore

plants

are

presumably

able

to

constantly

supply

co-transport

molecules

for

secondary

active

transporters

independently

of

environmental

conditions

The

existence

of

vacuoles

gives

plant

cells

more

self-sufficiency

than

animal

cells

and

explains

the

evolution

of

membrane

transport

genes

for

individual

cell

homeostasis

in

plants

Therefore

pump

and

secondary

transporter

systems

in

plants

are

more

divergent

than

in

animals

Fig.

of

membrane

transporter

proteins

of

each

class

Membrane

transporter

proteins

were

categorized

into

three

classes

-LRB-

ATP-dependent

-LSB-

pump

-RSB-

channel

and

secondary

transporter

-RRB-

and

compared

among

Escherichia

coli

Arabidopsis

thaliana

Oryza

sativa

Caenorhabditis

elegans

Drosophila

melanogaster

Homo

sapiens

NCBI

Neurospora

crassa

and

Saccharomyces

cerevisiae

analysis

of

membrane

transporter

gene

in

many

organismsGene

familyE

coli

thalianaO

sativaC

elegansD

melanogasterH

sapiens

N.

crassa

cerevisiae

MaterialPresent

inEnergy-dependent

-LRB-

pump

-RRB-

ABC

-LRB-

catalytic

-RRB-

AllH

PPase

P

a

-LRB-

vacuolar

-RRB-

IISP

-LRB-

mitochondria

-RRB-

P-ATPase

etc.

-LRB-

-RRB-

A

-LRB-

neuron

-RRB-

Annexin

-LRB-

mammal

-RRB-

Bestrophin

-LRB-

Cl

-RRB-

A

P

F

B

-LRB-

G

-RRB-

A

-LRB-

B-lymphocyte

-RRB-

ClC

AllConnexin

-LRB-

vertebrate

-RRB-

CSC

-LRB-

chloroplast

-RRB-

CytB

AllE-ClC

A

-LRB-

mammal

-RRB-

P

-LRB-

distant

homolog

-RRB-

A

-LRB-

epithelial

cell

brain

-RRB-

GIC

-LRB-

K

-RRB-

A

-LRB-

mammal

-RRB-

ICln

-LRB-

Cl

-RRB-

A

P

-LRB-

distant

homolog

-RRB-

Innexin

-LRB-

invertebrate

-RRB-

A

BLIC

-LRB-

-RRB-

-LRB-

-RRB-

Y

a

BMscL

Y

APCC

-LRB-

K

-RRB-

-LRB-

mammal

-RRB-

RIR-CaC

A

Y

-LRB-

vertebrate

-RRB-

B

VIC

or

AllPhosphotransferase

System

-LRB-

PTS

-RRB-

GPTS

TransporterAAA

-LRB-

chloroplast

-RRB-

AAAP

acid

AuxinA

P

F

Alanine

Antimonite

Y

BAE

H

Cl

-LRB-

P

Y

-RRB-

A

P

YAEC

Y

BAGCS

GlycineB

acid

PolyamineAllArsB

Antimonite

All

-LRB-

with

distant

homolog

-RRB-

BASS

acidAllBCCT

aBenE

AllCCC

K

Cl

AllCDF

or

etc.

F

Y

H+A

K+A

Amino

acidA

BDASS

P

Y

BDcu

B

-LRB-

G

-RRB-

DcuC

B

-LRB-

G

-RRB-

DMT

AllENT

P

YESS

BiopterinP

B

aFNT

NitrateY

B

-LRB-

E.

coli

Bacillus

-RRB-

GPH

P

F

a

BGUP

P

Y

F

BHAAAP

acidB

P

-LRB-

distant

homolog

-RRB-

KDGT

P

F

P

Glycolate

B

acid

-LRB-

L

I

V

-RRB-

B

aLIVCS

acid

-LRB-

L

I

V

-RRB-

BLysE

EukaryoteMET

etc.AMFS

-LRB-

Drugs

etc.

-RRB-

AllMTC

-LRB-

Anionic

substrate

-RRB-

Thiamine

P

F

Y

B

etc.AllNhaA

H+P

H+A

llNhaD

-LRB-

Li

-RRB-

H+P

BNiCoT

-LRB-

etc.

-RRB-

AllNSS

etc.

AOAT

P

F

B

aOST

compound

-LRB-

with

distant

homolog

-RRB-

AllPnaS

phosphate

A

-LRB-

mammal

-RRB-

POT

TiamineARhtB

acidB

aRND

AllSSS

AllTat

-LRB-

G

-RRB-

TDT

All

-LRB-

with

distant

homolog

-RRB-

ThrE

Serine

All

-LRB-

with

distant

homolog

-RRB-

TRAP-T

aTrk

P

Y

BZIP

Acid

Y

AllFeoB

B

aFeT

-LRB-

-RRB-

YFP

A

-LRB-

mammal

-RRB-

LPI

Y

BPnuC

mononucleotideBPPI

F

BPUP

Fatty

acidP

BNote

Present

in

A

animal

P

plant

F

fungi

Y

yeast

B

bacteria

a

archea

G

gramm

minus

bacteria

Energy

-LRB-

ATP

pyrophosphate

-RRB-

dependent

-LRB-

pump

-RRB-

system

We

compared

the

ATP-dependent

transport

genes

in

this

diverse

set

of

organisms

-LRB-

Table

Fig.

and

Supplemental

-RRB-

The

main

roles

of

the

ATP-dependent

-LRB-

pump

-RRB-

proteins

are

-LRB-

-RRB-

to

transport

molecules

in

specific

directions

independently

of

the

environmental

situation

and

-LRB-

-RRB-

to

transport

ions

to

form

a

concentration

gradient

between

the

areas

outside

and

inside

the

membrane

-LRB-

active

transport

-RRB-

Because

bacteria

can

not

control

the

concentrations

of

ions

or

metabolites

outside

the

cell

their

pumps

work

mainly

to

transport

molecules

In

E.

coli

most

of

the

ATP-dependent

genes

-LRB-

-RRB-

encode

ATP-binding

cassette

-LRB-

ABC

-RRB-

proteins

and

some

of

them

are

reported

to

encode

channel

proteins

-LRB-

Fig.

Table

-RRB-

-LRB-

Holland

et

al.

-RRB-

The

ABC

proteins

transport

various

substances

-LRB-

e.g.

ions

peptides

nucleosides

amino

acids

carbohydrates

proteins

-RRB-

ATP-dependently

in

all

organisms

-LRB-

Kolukisaoglu

et

al.

Garcia

et

al.

-RRB-

In

eukaryotes

many

functional

units

are

present

within

one

polypeptide

whereas

many

bacterial

ABC

subunits

are

encoded

by

individual

genes

This

results

in

an

inverted

relationship

between

prokaryotes

-LRB-

E.

coli

-RRB-

-LRB-

-RRB-

and

primitive

eukaryotes

-LRB-

yeast

-RRB-

-LRB-

-RRB-

Many

bacterial

ABC

proteins

are

located

in

the

plasma

membrane

and

serve

as

the

main

forces

in

energy-consuming

transport

for

both

import

and

export

of

substances

In

higher

eukaryotes

ABC

proteins

tend

to

export

substances

rather

than

function

in

import

reactions.Fig

of

numbers

of

pump

genes

among

various

organisms

Pump

gene

numbers

were

compared

among

Escherichia

coli

Arabidopsis

thaliana

Oryza

sativa

Caenorhabditis

elegans

Drosophila

melanogaster

Homo

sapiens

NCBI

Neurospora

crassa

and

Saccharomyces

cerevisiae

ABC

ATP-binding

Cassette

ArsAB

Arsenite-antimonite

Efflux

F-ATPase

H

or

translocating

F-type

V-type

and

A-type

ATPase

H

Ppase

H

translocating

Pyrophosphatase

IISP

General

Secretory

Pathway

-LRB-

Sec

-RRB-

MPT

Mitochondrial

Protein

Translocase

P-ATPase

P-type

ATPase

Structural

analyses

revealed

that

ABC

proteins

could

be

classified

as

half

size

-LRB-

homo

or

heterodimer

functional

-RRB-

or

full

size

-LRB-

monomer

functional

-RRB-

in

accordance

with

their

construction

The

number

of

genes

encoding

ABC

proteins

in

animals

is

the

same

as

or

slightly

less

than

in

bacteria

most

of

them

representing

whole

subunits

of

ABC

proteins

In

contrast

the

number

of

ABC

genes

in

plants

is

about

twice

that

in

animals

and

bacteria

primarily

due

to

the

encoding

of

half-size

proteins

-LRB-

Table

Fig.

and

Supplemental

-RRB-

-LRB-

Sanchez-Fernandez

et

al.

Sugiyama

et

al.

-RRB-

This

increase

in

numbers

of

genes

encoding

half-size

-LRB-

e.g.

WBC

-RRB-

and

full-size

-LRB-

PDR

pleiotropic

drug

resistance

-RRB-

proteins

supposedly

permits

stage

and

tissue-specific

regulation

of

various

substances

-LRB-

including

plant-specific

substances

-RRB-

Therefore

increases

in

the

capacity

of

import

and

export

transport

systems

by

an

increase

in

the

number

of

half-size

ABC

proteins

have

allowed

plants

to

fulfill

their

unique

roles

In

the

import

and

efflux

of

ions

and

the

creation

of

ion

gradients

for

secondary

transport

P-type

ATPases

transport

many

species

of

ion

-LRB-

e.g.

H

K

-RRB-

in

both

directions

in

the

cell

-LRB-

Table

Fig.

and

Supplementary

-RRB-

-LRB-

Axelsen

and

Palmgren

Baxter

et

al.

-RRB-

Control

of

ion

concentrations

outside

bacterial

cells

is

not

possible

Only

a

few

of

the

bacterial

genes

involved

in

systems

for

the

uptake

or

efflux

of

ions

-LRB-

uptake

K

efflux

Ag

uptake

or

efflux

depending

on

the

system

-RRB-

have

been

characterized

and

each

of

the

enzymes

encoded

comprises

a

distinct

subfamily

-LRB-

Banci

et

al.

-RRB-

Eukaryotes

have

diverged

in

terms

of

their

transport

substances

and

have

also

adapted

genes

for

making

the

ion

gradients

for

secondary

transport

One

of

the

most

important

ATPases

in

animals

K+ATP

ase

does

not

exist

in

plants

K+ATP

ase

makes

a

ion

gradient

across

the

plasma

membrane

and

forms

the

basis

of

membrane

voltage

and

secondary

active

transport

in

animals

On

the

other

hand

plants

use

H

gradients

for

secondary

transport

and

more

than

isoforms

and

vacuole-type

H+ATP

ases

are

involved

in

stage

and

tissue-specific

control

This

difference

in

adapted

ion

gradients

reflects

the

ion

concentrations

in

the

cells

and

the

demand

for

nutrient

ions

A

constant

supply

of

ions

is

required

to

retain

homeostasis

of

animal

cells

and

rapid

signal

transmission

systems

-LRB-

e.g.

muscles

nerves

-RRB-

In

contrast

plants

use

the

more

abundant

H

ion

to

make

cation

gradients

and

have

special

systems

for

transporting

H

ions

into

vacuolar

pools

For

transporting

H

into

vacuoles

higher

plants

have

a

vacuole-type

ATPase

and

an

H

translocating

pyrophosphatase

-LRB-

H

PPase

-RRB-

-LRB-

Table

Fig.

and

Supplementary

-RRB-

that

are

comparable

to

bacterial

ATPase

and

PPase

respectively

-LRB-

Sivula

et

al.

Sze

et

al.

-RRB-

Therefore

plants

presumably

adapted

bacterial

H

ATPase

systems

to

help

in

H

ion

concentration

steps

whereas

animals

developed

completely

new

systems

to

make

ion

gradients

Ion

channel

systems

Ion

channels

are

the

gates

in

the

membranes

that

open

or

close

in

response

to

signals

such

as

mechanical

or

electrical

stimulation

and

ligand

binding

Therefore

ion

channels

are

closely

involved

in

determining

whether

or

not

ionic

gradients

are

available

Multicellular

organisms

can

control

the

ion

concentrations

in

the

tissues

on

both

sides

of

the

cell

membrane

whereas

monocellular

organisms

usually

find

it

hard

to

control

the

ion

gradient

outside

the

cell

Thus

the

use

of

ion

channels

becomes

restrictive

and

unidirectional

in

these

more

primitive

organisms

Unlike

in

eukaryotes

the

channel

system

in

prokaryotes

is

not

well

adapted

to

transport

-LRB-

Table

Fig.

and

Supplemental

-RRB-

Of

the

total

number

of

genes

encoding

membrane

transport

proteins

in

prokaryotes

fewer

than

are

channel

genes

and

they

mainly

regulate

osmotic

homeostasis

in

the

cell

Ions

-LRB-

e.g.

Cl

K

metal

-RRB-

water

and

osmolytes

are

imported

or

exported

by

the

channels

in

their

restricted

role

In

contrast

animals

can

make

various

ion

gradients

precisely

and

in

particular

can

develop

channel

systems

very

well.Fig

of

numbers

of

channel

genes

among

various

organisms

Channel

gene

numbers

were

compared

among

many

organisms

-LRB-

E.

coli

A.

thaliana

O.

sativa

C.

elegans

D.

melanogaster

H.

sapiens

NCBI

N.

crassa

and

S.

cerevisiae

-RRB-

ACC

ATP-gated

Cation

Channel

Bestrophin

Anion

Channel-forming

Bestrophin

Channel

ClC

Chloride

Channel

Connexin

Gap

Junction-forming

Connexin

CytB

Phagocyte

NADPH

Oxidase-associated

Cytochrome

-LRB-

CytB

-RRB-

H

channel

E-ClC

Epithelial

Chloride

Channel

EnaC

Epithelial

Channel

GIC

Glutamate-gated

Ion

Channel

Cation

Channel-forming

Heat

Shock

ICC

Intracellular

Chloride

Channel

Icln

Nucleotide-sensitive

Anion-selective

Channel

Innexin

Gap

Junction-forming

Innexin

IRK-C

Inward

Rectifier

K

Channel

LIC

Ligand-gated

Ion

Channel

of

Neurotransmitter

Receptors

Yeast

Stretch-Activated

Cation-Selective

Channel

MIP

Major

Intrinsic

Protein

MIT

CorA

Metal

Ion

Transporter

MscL

Large

Conductance

Mechanosensitive

Ion

Channel

MscS

Small

Conductance

Mechanosensitive

Ion

Channel

Non-selective

Cation

O-ClC

Organellar

Chloride

Channel

PCC

Polycystin

Cation

Channel

PLM

Phospholemman

RIR-CaC

Ryanodine-Inositol

Receptor

Channel

Chloroplast

Envelope

Anion

Channel-forming

TRP-CC

Transient

Receptor

Potential

Channel

UT

Urea

Transporter

VIC

Voltage-gated

Ion

Channel

Compared

with

other

systems

the

channel

system

is

the

fastest

at

transporting

molecules

without

consuming

energy

-LRB-

i.e.

when

it

is

not

necessary

to

transport

against

electrical

gradients

-RRB-

Therefore

animals

-LRB-

especially

vertebrates

-RRB-

have

adapted

these

systems

to

nerve

and

muscle

signal

transmission

Nervous-system-specific

channels

such

as

neurotransmitter-responsible

channels

-LRB-

connexin

ENaC

innexin

LIC

RIR-CaC

TRP-CC

-RRB-

are

found

only

in

animals

The

numbers

of

channels

that

are

membrane-voltage-dependent

for

K

and

ion

transport

are

dramatically

increased

in

both

vertebrates

and

invertebrates

-LRB-

Table

Fig.

Sheng

et

al.

Du

et

al.

Clapham

Hua

et

al.

Miyazawa

et

al.

-RRB-

Because

animals

form

and

K

gradients

with

the

K

ATPase

pump

system

there

is

also

an

increase

in

the

numbers

of

species

of

and

K

ion

channels

in

animals

Thus

the

total

number

of

channel

genes

in

humans

-LRB-

-RRB-

is

about

twice

that

in

plants

-LRB-

-RRB-

-LRB-

Table

Fig.

-RRB-

This

difference

is

supposedly

related

to

the

ecological

and

physiological

specificity

of

plants

Sometimes

constant

acquisition

of

unevenly

distributed

resources

is

difficult

for

plants

Additionally

the

ion

concentrations

in

the

vascular

systems

and

intercellular

spaces

of

plants

unlike

those

in

animals

are

difficult

to

control

precisely

Therefore

some

of

the

ion

channels

and

membrane-voltage-dependent

channels

are

not

as

divergent

as

in

animals

Although

plants

lack

neurotransmission

systems

and

have

fewer

membrane-voltage-dependent

systems

that

use

and

K

ion

channels

the

channel

genes

for

ion

homeostasis

and

signal

transduction

-LRB-

e.g.

ClC

GIC

-RRB-

are

as

well

developed

as

in

animals

and

the

numbers

of

some

channel

genes

-LRB-

CytB

and

MscS

-RRB-

are

in

fact

specifically

increased

in

plants

-LRB-

Table

Fig.

-RRB-

Table

of

the

genome

size

total

and

membrane

transport

gene

numbers

in

many

organismsE

coli

thalianaO

sativaC

elegansD

melanogasterH

sapiens

N.

crassa

cerevisiae

Genome

Size

-LRB-

Mb

-RRB-

Total

gene

Total

Transporter

per

Mb

Transporters

per

whole

gene0.0820.0380.0330.0330.0440.0250.0340.052ATP-dependent

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Ion

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Phosphotransferase

Systems

-LRB-

PTS

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Comparison

of

protein

structures

indicates

that

the

fundamental

structures

of

ion

channels

-LRB-

numbers

of

transmembrane

domains

pore-forming

helices

-RRB-

are

common

in

many

organisms

but

local

similarities

in

individual

regions

among

organisms

are

low

-LRB-

especially

in

the

N

and

C

terminus

fragments

-RRB-

Some

of

the

genes

-LRB-

e.g.

for

shaker-type

K

channel

SKOR-type

K

channel

-RRB-

have

low

levels

of

similarity

among

the

whole

structures

of

plants

and

animals

-LRB-

Supplemental

-RRB-

In

contrast

the

major

intrinsic

protein

-LRB-

MIP

-RRB-

gene

family

which

encodes

water-transport

proteins

is

specifically

well

developed

in

plants

-LRB-

Zardoya

-RRB-

The

numbers

of

these

genes

in

tissues

and

at

different

stages

are

times

those

in

animals

-LRB-

vs.

-RRB-

-LRB-

Table

Fig.

-RRB-

-LRB-

Quigley

et

al.

Sakurai

et

al.

-RRB-

MIPs

are

abundant

in

the

plasma

membrane

-LRB-

of

total

membrane

protein

-RRB-

and

vacuoles

-LRB-

of

total

membrane

protein

-RRB-

of

plants

therefore

a

high

level

of

water

transport

is

carried

out

at

plant

cell

membranes

The

importance

of

the

acquisition

of

water

has

presumably

resulted

in

the

diversification

and

development

of

water

channels

in

plants

Plants

also

use

water

pressure

for

regulation

of

movement

-LRB-

e.g.

stomatal

opening

leaf

and

petal

angle

changes

-RRB-

and

for

transmitting

signals

in

various

homeostatic

functions

Therefore

plants

have

presumably

developed

signal

transduction

systems

that

rely

on

water

molecules

instead

of

the

neurotransmitters

and

membrane

voltage

changes

used

in

animals

Structural

analyses

indicate

divergence

in

the

level

of

conservation

of

MIP

subfamilies

-LRB-

PIP

TIP

SIP

NIP

AQP

and

GLP

-RRB-

-LRB-

Supplemental

-RRB-

The

plasma-membrane-intrinsic

proteins

-LRB-

PIPs

-RRB-

are

well

conserved

among

organisms

but

animal

-LRB-

AQP

-RRB-

and

plant-specific

-LRB-

SIP

NIP

TIP

-RRB-

genes

have

diverged

and

closely

related

channel

proteins

have

low

levels

of

similarity

within

each

organism

-LRB-

Supplementary

-RRB-

This

also

suggests

a

specific

diversification

of

the

water

transport

genes

in

plants

The

structures

of

the

channels

in

plants

are

simple

compared

with

those

in

animals

-LRB-

Supplementary

-RRB-

A

decrease

in

the

membrane

spanning

times

and

in

the

number

of

subunits

of

working

systems

can

be

detected

in

many

channels

-LRB-

e.g.

VIC

-LSB-

VDCC

-RSB-

CIC

-RRB-

-LRB-

Supplementary

-RRB-

Therefore

animals

have

developed

channel

systems

especially

for

the

exquisite

control

of

ions

membrane

voltages

and

the

signal

transduction

pathways

of

specific

ligands

-LRB-

e.g.

neurotransmitters

-RRB-

Phosphotransferase

system

-LRB-

bacteria

-RRB-

Bacteria

have

specific

membrane

transport

systems

-LRB-

phosphotransferase

systems

-RRB-

for

carbohydrates

-LRB-

sugar

-RRB-

and

phosphates

-LRB-

Tables

-RRB-

-LRB-

Barabote

and

Saier

-RRB-

These

systems

use

phosphate

phosphoenol

pyruvate

-LRB-

PEP

-RRB-

as

the

energy

source

for

phosphorylation

and

for

transport

of

carbohydrates

with

the

aid

of

enzyme

complexes

Carbohydrates

-LRB-

e.g.

glucose

fructose

mannitol

sorbitol

-RRB-

are

transported

against

a

concentration

gradient

with

concomitant

phosphorylation

PEP

is

transferred

via

the

soluble

-LRB-

and

non-sugar-specific

-RRB-

enzymes

EI

and

HPr

to

the

enzyme

complex

EII

EII

is

made

up

of

components

A

B

and

C

which

depending

on

the

sugar

specificity

and

bacterium

involved

may

be

the

domains

of

composite

proteins

component/domain

C

is

a

permease

and

is

anchored

to

the

cytoplasmic

membrane

Because

the

amount

of

phosphorylation

of

the

enzymes

influences

other

regulatory

mechanisms

in

the

cells

-LRB-

e.g.

catabolite

repression

chemotaxis

-RRB-

the

whole

cell

needs

a

multiple-component

complex

enzymatic

control

system

Therefore

the

larger

and

more

complicated

cells

of

animals

and

plants

have

not

adapted

this

system

and

instead

use

ATP-dependent

and

secondary

transporter

systems

to

transport

carbohydrates

Secondary

transporter

-LRB-

transporter

-RRB-

system

The

secondary

transporter

system

works

via

the

concentration

gradient

of

co-transporter

molecules

It

is

efficient

to

use

a

few

abundant

molecules

as

common

co-transporter

molecules

for

various

transport

substrates

Therefore

in

accordance

with

the

species

of

ion

for

which

there

is

a

gradient

between

the

inside

and

outside

of

the

cell

major

co-transporter

molecules

were

selected

and

secondary

transporter

systems

developed

for

them

There

are

more

than

species

of

gene

families

in

the

secondary

transporter

systems

of

all

organisms

and

various

substances

are

transported

Compared

with

those

in

the

pump

and

channel

systems

the

genes

involved

in

the

secondary

transporter

system

are

most

divergent

in

plants

and

bacteria

in

animals

there

are

about

of

these

genes

-LRB-

of

the

total

-RRB-

-LRB-

Tables

Fig.

-RRB-

Fig.

of

numbers

of

secondary

transport

genes

among

organisms

Secondary

transporter

gene

numbers

were

compared

among

Escherichia

coli

Arabidopsis

thaliana

Oryza

sativa

Caenorhabditis

elegans

Drosophila

melanogaster

Homo

sapiens

NCBI

Neurospora

crassa

and

Saccharomyces

cerevisiae

AAAP

Amino

Acid/Auxin

Permease

AE

Anion

Exchanger

AEC

Auxin

Efflux

Carrier

Amt

Ammonium

or

Ammonia

Transporter

APC

Amino

Acid-Polyamine-Organocation

BASS

Bile

Acid

Symporter

BCCT

Betaine/Carnitine/Choline

Transporter

BenE

Benzoate

H

Symporter

CaCA

Cation

Antiporter

CCC

Cation-Chloride

Cotransporter

CDF

Cation

Diffusion

Facilitator

CNT

Concentrative

Nucleoside

Transporter

Monovalent

Cation

Proton

Monovalent

Cation

Proton

DAACS

Dicarboxylate/Amino

Acid

Cation

-LRB-

or

H

-RRB-

Symporter

DASS

Divalent

Anion

Symporter

DcuC

Uptake

C

DMT

Drug/Metabolite

Transporter

ENT

Equilibrative

Nucleoside

Transporter

FBT

Folate-Biopterin

Transporter

GntP

Gluconate

H

Symporter

GPH

Glycoside-Pentoside-Hexuronide

-LRB-

GPH

-RRB-

Cation

Symporter

HAAAP

Hydroxy/Aromatic

Amino

Acid

Permease

KUP

K

Uptake

Permease

LCT

Lysosomal

Cystine

Transporter

MC

Mitochondrial

Carrier

MET

TMS

Multidrug

Endosomal

Transporter

MFS

Major

Facilitator

Superfamily

MOP

Multidrug/Oligosaccharidyl-lipid/Polysaccharide

Flippase

Superfamily

Nucleobase

Cation

Nramp

Metal

Ion

-LRB-

iron

-RRB-

Transporter

NSS

Neurotransmitter

Sodium

Symporter

OAT

Organo

Anion

Transporter

OPT

Oligopeptide

Transporter

PnaS

Phosphate

Symporter

POT

Proton-dependent

Oligopeptide

Transporter

RFC

Reduced

Folate

Carrier

RhtB

Resistance

to

Homoserine/Threonine

RND

Resistance-Nodulation-Cell

Division

SSS

Solute

Sodium

Symporter

SulP

Sulfate

Permease

TDT

Tellurite-resistance/Dicarboxylate

Transporter

Trk

K

Transporter

ZIP

Zinc

-LRB-

-RRB-

Iron

-LRB-

-RRB-

Permease

Organism-specific

gene

families

in

bacteria

include

those

encoding

proteins

-LRB-

e.g.

APC

HAAAP

-RRB-

that

transport

many

species

of

amino

acid

or

carbohydrate

-LRB-

GntP

Dcu

-RRB-

or

other

substances

-LRB-

BCCT

BenE

-RRB-

-LRB-

Neidle

et

al.

Golby

et

al.

Saier

et

al.

Jack

et

al.

Samsonov

et

al.

Prakash

et

al.

-RRB-

Gene

families

in

animals

include

genes

for

specific

transport

proteins

-LRB-

NSS

MET

-RRB-

that

carry

neurotransmitters

and

hydrophobic

compounds

-LRB-

Beckman

and

Quick

Hogue

et

al.

Yamashita

et

al.

-RRB-

Those

in

plants

include

genes

with

dramatically

increased

expression

encoding

proteins

-LRB-

AAAP

DMT

MFS

OPT

POT

-RRB-

that

transport

such

substances

as

amino

acids

cations

and

carbohydrates

-LRB-

Fig.

-RRB-

-LRB-

Fischer

et

al.

Pao

et

al.

Saier

et

al.

Jack

et

al.

Koh

et

al.

Wipf

et

al.

-RRB-

The

greater

number

of

secondary

active

transporters

in

plants

is

supposedly

due

to

the

existence

of

vacuoles

Secondary

active

transporters

need

an

ion

gradient

across

the

membranes

and

vacuoles

can

pool

many

substances

at

high

concentrations

and

supply

co-transporter

molecules

instantly

at

any

time

Therefore

the

secondary

transporter

systems

easily

control

transport

and

have

diverged

more

in

plants

than

in

animals

The

major

facilitator

superfamily

-LRB-

MFS

-RRB-

is

the

largest

secondary

transporter

family

in

whole

organisms

its

members

transport

various

substances

-LRB-

e.g.

carbohydrates

phosphates

amino

acids

cations

-RRB-

with

or

H

ions

-LRB-

Pao

et

al.

Burckhardt

and

Wolff

Lemoine

-RRB-

There

are

many

modifications

of

these

superfamily

genes

among

organisms

Bacteria

have

amino

acid

and

drug

efflux

families

animals

have

neurotransmitter

transport

families

and

plants

have

diverse

carbohydrate-transport

genes

-LRB-

Tables

Fig.

and

Supplementary

-RRB-

The

proteins

have

structures

typical

of

secondary

active

transporters

transmembrane

domains

co-transporter

molecules

and

material-binding

domains

Comparative

analyses

indicate

that

their

fundamental

structure

membrane-spanning

pore-forming

and

substrate-binding

sites

is

conserved

but

similarities

between

individual

domains

are

generally

low

among

organisms

-LRB-

Tables

Fig.

and

Supplementary

-RRB-

The

transporter

proteins

are

encoded

by

single

transcription

units

and

work

as

monomers

Therefore

this

transport

system

is

easy

to

adapt

to

new

purposes

than

are

the

higher

pump

and

ion

channel

systems

Although

the

secondary

transporter

system

can

adapt

to

many

kinds

of

concentration

gradients

generally

few

ion

species

are

used

Most

of

the

secondary

active

transporters

depend

on

two

ions

and

H

In

the

whole

organism

more

than

of

the

genes

depend

on

these

ions

as

co-transporter

molecules

-LRB-

Table

Fig.

-RRB-

However

in

many

cases

each

ion

can

be

substituted

animals

use

ions

as

co-transporter

molecules

and

bacteria

and

plants

use

H

ions

This

has

caused

animals

to

make

a

ion

gradient

with

K

ATPase

for

membrane

voltage

and

plants

and

bacteria

to

make

H

ion

gradients

with

P-type

ATPase

and

H

PPase

respectively

The

transporter

genes

of

animals

and

plants

have

specifically

adapted

to

and

H

co-transporter

systems

-LRB-

Table

Fig.

-RRB-

Therefore

both

animals

and

plants

have

developed

transporter

genes

adapted

to

ion

gradients.Table

of

secondary

transporter

genesE

coli

A.

thalianaO

sativaC

elegansD

melanogasterH

sapiens

N.

crassa

S.

cerevisiae

Present

inMaterialCotransporterDirectionAAA

-LRB-

chloroplast

-RRB-

ATPH

in

-LRB-

same

-RRB-

P

F

YAmino

acid

AuxinH

in

-LRB-

same

-RRB-

AAE

l-Alanine

each

otherboth

-LRB-

anti

-RRB-

H

in

-LRB-

same

-RRB-

YArsenite

Antimonite

outAE

P

YNa

H

Cr

Cl

both

-LRB-

anti

-RRB-

AEC

Y

BAuxinH

out

-LRB-

anti

-RRB-

BAlanine

GycineNa

H

in

-LRB-

same

-RRB-

Amt

acid

PolyamineH

Solutein

-LRB-

same

-RRB-

Y

B

aArmate

acid

-LRB-

Malate

etc.

-RRB-

outArsB

-LRB-

with

distant

homolog

-RRB-

Arsenite

Antimonite

outBASS

acidNa

in

-LRB-

same

-RRB-

BCCT

aBetaine/Carnitine/CholineH

in

-LRB-

same

-RRB-

BenE

in

-LRB-

same

-RRB-

CaCA

H

both

-LRB-

anti

-RRB-

CCC

K

Cl

K

Cl

both

-LRB-

same

-RRB-

cation

-LRB-

-RRB-

H

K

out

-LRB-

antit

-RRB-

CHR

H

both

-LRB-

anti

-RRB-

Y

BNucleosideH

in

-LRB-

same

-RRB-

H+H

Cl

both

-LRB-

anti

-RRB-

K+H

-LRB-

or

itself

-RRB-

out

-LRB-

anti

-RRB-

DAACS

BDicarboxylate

Amino

acidH

in

-LRB-

same

-RRB-

P

Y

B

-LRB-

G

-RRB-

Aminoacid

Sulfate

Phosphate

etc.

H

in

-LRB-

same

-RRB-

both

-LRB-

anti

-RRB-

-LRB-

G

-RRB-

Dicarboxylateboth

-LRB-

anti

-RRB-

-LRB-

G

-RRB-

Dicarboxylatein

-LRB-

same

H

-RRB-

both

-LRB-

anti

-RRB-

DMT

Carbohydrate

Sugar

Nucleotide

etc.

H

Nucleotideout

-LRB-

anti

-RRB-

P

YNucleoside?bothESS

in

-LRB-

same

-RRB-

FBT

a

BFolate

BiopterinH

in

-LRB-

same

-RRB-

FNT

a

BFormate

NitrateH

in

-LRB-

same

-RRB-

GntP

-LRB-

E.

coli

Bacillus

-RRB-

CarbohydrateH

in

-LRB-

same

-RRB-

P

F

a

BSugarH

Li

in

-LRB-

same

-RRB-

GUP

P

F

Y

BGlycerolH

in

-LRB-

same

-RRB-

HAAAP

P

-LRB-

distant

homolog

-RRB-

Amino

acidH

in

-LRB-

same

-RRB-

KDGT

in

-LRB-

same

-RRB-

KUP

F

BK

P

FCystineH

in

-LRB-

same

-RRB-

LctP

BLactate

Glycolate

H

in

-LRB-

same

-RRB-

BAmino

acid

-LRB-

L

I

V

-RRB-

H

in

-LRB-

same

-RRB-

acid

-LRB-

L

I

V

-RRB-

H

in

-LRB-

same

-RRB-

LysE

OH

out

-LRB-

OH

same

H

anti

-RRB-

MC

Carbohydrate

Sugar

Nucleotide

etc.

Variousboth

-LRB-

anti

-RRB-

Hydrophobic

compound

H

both

-LRB-

anti

-RRB-

MFS

Carbohydrate

Sugar

etc.

H

Variousboth

-LRB-

H

same

others

both

-RRB-

MOP

-LRB-

MATE

-RRB-

-LRB-

Drugs

Polysaccharides

etc.

-RRB-

-LRB-

H

-RRB-

out

-LRB-

anti

-RRB-

MTC

-LRB-

Anionic

substrate

-RRB-

H

in

-LRB-

same

-RRB-

F

Y

a

BNucleobase

Thiamine

H

in

-LRB-

same

-RRB-

-LRB-

Ascorbate

-LRB-

mouse

only

-RRB-

-RRB-

H

-LRB-

-LRB-

Ascotbate

-RRB-

-RRB-

in

-LRB-

same

-RRB-

NhaA

H+N

a

H

both

-LRB-

anti

-RRB-

NhaB

H+N

a

H

both

-LRB-

anti

-RRB-

NhaD

BNa

-LRB-

Li

-RRB-

H+N

a

-LRB-

Li

-RRB-

H

both

-LRB-

anti

-RRB-

NiCoT

both

-LRB-

anti

-RRB-

Nramp

etc.

H

in

-LRB-

same

-RRB-

NSS

Amino

acids

Osmolytes

Cl

etc.

in

-LRB-

same

-RRB-

OAT

P

F

YVariousAnion

both

-LRB-

anti

-RRB-

OPT

B

aOligopeptideH

in

-LRB-

same

-RRB-

compound

-LRB-

mostly

anions

-RRB-

each

otherboth

-LRB-

anti

-RRB-

-LRB-

with

distant

homolog

-RRB-

Proteineach

otherboth

-LRB-

anti

-RRB-

PiT

H

in

-LRB-

same

-RRB-

PNaS

B

-LRB-

distant

homolog

-RRB-

Inorganic

phosphate

in

-LRB-

same

-RRB-

POT

both

-LRB-

same

-RRB-

TiamineH

OH

anionboth

-LRB-

anti

-RRB-

BAmino

acidH

out

-LRB-

anti

-RRB-

metals

Drugs

Lipids

etc.

H

out

-LRB-

anti

-RRB-

Amino

acid

Organo

cation

AnionNa

-LRB-

H

can

replace

but

reduces

affinity

-RRB-

in

-LRB-

same

-RRB-

SulP

H

in

-LRB-

same

-RRB-

-LRB-

G

-RRB-

P

-LRB-

distant

homolog

-RRB-

ProteinnoneoutTDT

-LRB-

with

distant

homolog

-RRB-

Tellurite

H

in

-LRB-

same

-RRB-

-LRB-

with

distant

homolog

-RRB-

Threonine

Serine

H

both

-LRB-

anti

-RRB-

BVariousH

in

-LRB-

same

-RRB-

Trk

Y

BK+H

in

-LRB-

same

-RRB-

ZIP

noneinNote

Present

in

A

animal

P

plant

F

fungi

Y

yeast

B

bacteria

a

archea

G

gramm

minus

bacteria

Direction

in

out

and

both

indicate

transport

direction

of

materials

through

cell

membrane

and

same

means

material

and

cotranspoter

move

to

the

same

dirction

anti

means

the

different

directionsFig

of

co-transport

molecules

of

secondary

active

transporters

among

various

organisms

Numbers

of

co-transport

molecules

of

secondary

active

transporters

were

compared

among

Escherichia

coli

Arabidopsis

thaliana

Oryza

sativa

Caenorhabditis

elegans

Drosophila

melanogaster

Homo

sapiens

NCBI

Neurospora

crassa

and

Saccharomyces

cerevisiae

In

animals

many

molecules

are

transported

with

different

co-transporter

molecules

Examples

are

-LRB-

-RRB-

molecules

-LRB-

e.g.

neurotransmitters

organic

anions

phosphates

-RRB-

transported

with

ion

-LRB-

NSS

BASS

Pnas

-RRB-

-LRB-

Beckman

and

Quick

Saier

et

al.

Kramer

et

al.

Segawa

et

al.

Radchenko

et

al.

-RRB-

-LRB-

-RRB-

molecules

-LRB-

e.g.

amino

acids

nucleosides

-RRB-

transported

with

H

ions

-LRB-

e.g.

AAAP

APC

DMT

LCT

MET

-RRB-

-LRB-

Steiner

et

al.

Jack

et

al.

Zhai

et

al.

Gasol

et

al.

-RRB-

-LRB-

-RRB-

molecules

-LRB-

e.g.

anions

cations

sugars

nucleosides

-RRB-

transported

with

either

or

H

ions

-LRB-

e.g.

AE

CaCA

DAACS

GPH

CNT

-RRB-

-LRB-

Reinders

and

Ward

Ritzel

et

al.

Zhu

et

al.

Cai

and

Lytton

Ryan

et

al.

-RRB-

and

-LRB-

-RRB-

others

-LRB-

e.g.

CCC

OAT

RFC

-RRB-

transported

with

other

molecules

-LRB-

Russell

Flintoff

et

al.

Hagenbuch

and

Meier

-RRB-

In

contrast

in

plants

almost

all

substances

-LRB-

K

KUP

N

-LRB-

-RRB-

Amt

phosphate

PiT

Pht

-LSB-

MFS

-RSB-

and

TPT

-LSB-

DMT

-RSB-

SulP

sucrose

GPH

sugar

alcohol

SAT

-LSB-

MFS

-RSB-

monosaccharide

MST

-LSB-

MFS

-RSB-

amino

acid

APC

and

AAAP

nucleosides

-RRB-

including

many

cations

anions

metals

and

drugs

are

co-transported

with

H

ions

Only

BASS

-LRB-

organic

acid

transporter

-RRB-

and

NhaD

-LRB-

H

antiporter

efflux

system

of

vacuole

-RRB-

use

ions

as

co-transporter

molecules

for

efflux

of

ions

from

the

cytoplasm

so

plants

do

not

require

extensive

sodium

uptake

-LRB-

Table

Fig.

-RRB-

-LRB-

Smith

et

al.

Naderi

and

Saier

Fu

and

Luan

Nozaki

et

al.

Pao

et

al.

Daram

et

al.

de

Koning

and

Diallinas

Jack

et

al.

Khademi

et

al.

-RRB-

Accumulation

of

H

ions

makes

solutions

acidic

and

prevents

many

enzymatic

and

biochemical

reactions

so

plants

pool

H

ions

in

vacuoles

inside

the

cytoplasm

thereby

establishing

a

stable

ion

gradient

for

transport

energy

Plants

make

H

gradients

not

only

inside

cells

but

also

outside

cells

by

the

H

pump

and

they

absorb

many

substances

by

secondary

transporter

systems

This

H

adapting

system

presumably

has

led

to

diversification

of

H

co-transport

in

secondary

transporter

systems

in

plants

Comparative

analysis

of

membrane

transporter

systems

in

Arabidopsis

and

rice

We

also

compared

the

numbers

of

membrane

transporter

genes

in

Arabidopsis

and

rice

The

rice

genome

size

-LRB-

Mb

-RRB-

is

more

than

three

times

that

of

Arabidopsis

-LRB-

Mb

-RRB-

but

the

total

number

of

membrane

transporter

proteins

-LRB-

-RRB-

is

only

times

that

in

Arabidopsis

-LRB-

-RRB-

-LRB-

Table

-RRB-

The

proportions

of

pump

channel

and

secondary

transporter

proteins

are

almost

the

same

and

the

numbers

of

genes

in

many

individual

gene

families

are

times

those

in

rice

However

Arabidopsis

has

more

multi-efflux

proteins

and

proteins

involved

in

secondary

active

transport

-LRB-

DMT

MOP

-RRB-

and

in

channel-type

signal

transduction

systems

-LRB-

CytB

GIC

-RRB-

-LRB-

Fig.

-RRB-

Arabidopsis

is

a

wild

plant

that

lives

in

diverse

soil

conditions

and

under

environmental

stress

whereas

rice

is

a

cultivated

crop

plant

grown

under

more

stable

environmental

conditions

Therefore

Arabidopsis

has

diverged

more

than

the

rice

cultivar

Nipponbare

to

form

transporter

systems

involved

in

multi-efflux

and

stress

response

signaling

On

the

other

hand

rice

has

more

pump

and

secondary

transporter

genes

-LRB-

ABC

P-type

ATPase

MFS

POT

-RRB-

for

carbohydrate

and

nutrient

transport

systems

-LRB-

Fig.

-RRB-

This

divergence

of

carbohydrate

transporters

might

have

been

influenced

by

artificial

selection

during

which

individuals

with

larger

seed

size

and

numbers

were

chosen

to

be

cultivated

Additionally

amino

acid

-LRB-

AAAP

-RRB-

ammonia

-LRB-

Amt

-RRB-

sulfate

-LRB-

SulP

-RRB-

metal

ion

-LRB-

ZIP

-RRB-

-LRB-

CaCA

-RRB-

and

K

transport

protein

gene

families

-LRB-

VIC

KUP

-RRB-

are

specifically

diverged

in

rice

This

may

have

been

caused

by

the

need

for

nutrient

supplements

for

rapid

growth

in

subtropical

plants

These

differences

may

also

result

from

dissimilarities

between

monocots

and

dicots

Although

the

cell

structures

are

the

same

in

both

plants

there

are

many

differences

in

the

basic

structure

of

tissues

and

organs

Differences

in

root

and

vascular

bundle

structure

may

be

related

to

differences

in

uptake

efficiency

of

nutrients

The

levels

of

uptake

and

transport

amounts

of

ions

such

as

K

and

other

nutrients

differ

between

monocots

and

dicots

this

might

be

related

to

differences

in

the

numbers

of

membrane

transport

genes

Fig.

of

membrane

transport

genes

in

Arabidopsis

and

rice

Numbers

of

the

membrane

transporter

gene

families

are

compared

in

Arabidopsis

and

rice

ABC

ATP-binding

Cassette

P-ATPase

P-type

ATPase

CytB

Phagocyte

NADPH

Oxidase-associated

Cytochrome

-LRB-

CytB

-RRB-

H

channel

GIC

Glutamate-gated

Ion

Channel

MIP

Major

Intrinsic

Protein

VIC

Voltage-gated

Ion

Channel

AAAP

Amino

Acid/Auxin

Permease

Amt

Ammonium

or

Ammonia

Transporter

CaCA

Cation

Antiporter

Monovalent

Cation

Proton

DMT

Drug/Metabolite

Transporter

KUP

K

Uptake

Permease

MFS

Major

Facilitator

Superfamily

MOP

Flippase

Superfamily

Nucleobase

Cation

OPT

Oligopeptide

Transporter

POT

Proton-dependent

Oligopeptide

Transporter

SulP

Sulfate

Permease

ZIP

Zinc

-LRB-

-RRB-

Iron

-LRB-

-RRB-

Permease

PUP

Peptide

Uptake

or

Activated

Fatty

Acid

Export

Permease

Comparison

of

the

membrane

transport

genes

in

Arabidopsis

and

rice

pointed

to

the

directions

of

evolution

of

these

plants

in

response

to

the

selection

pressures

of

their

differing

environmental

situations

The

level

of

conservation

of

gene

families

in

Arabidopsis

and

rice

varies

depending

on

the

category

Rice

membrane

transporters

were

orthologous

with

those

of

Arabidopsis

this

is

higher

than

the

ortholog

found

in

transcription

factors

-LRB-

Xiong

et

al.

-RRB-

The

levels

of

expression

of

membrane

transport

genes

are

limited

precisely

within

a

small

range

and

these

genes

are

critical

for

survival

This

may

be

the

reason

why

the

membrane

transport

genes

were

less

divergent

than

the

transcription

factors

Therefore

the

specifically

divergent

gene

families

in

Arabidopsis

and

rice

are

related

to

the

indispensable

and

unique

systems

in

each

plant

Divergence

of

gene

patterns

might

indicate

differences

between

wild

weeds

and

crop

cultivars

that

have

been

selected

for

growth

in

specific

and

possibly

new

environments

Comparison

of

the

overall

gene

compositions

of

bacteria

-LRB-

E.

coli

-RRB-

animals

-LRB-

H.

sapiens

-RRB-

and

plants

In

the

eight

diverse

organisms

that

we

compared

the

number

of

membrane

transporter

genes

-LRB-

-RRB-

varied

less

than

that

of

genes

in

other

categories

The

minimum

number

of

genes

indispensable

to

retaining

cell

membrane

transport

homeostasis

thus

seems

to

be

as

found

in

bacteria

yeasts

and

fungi

Many

of

the

additional

newly

diverged

genes

of

higher

animals

and

plants

are

channel

transporter

genes

and

secondary

active

transporters

that

facilitate

adaptation

to

fluctuating

concentration

gradients

present

in

their

environments

Moreover

many

newly

acquired

transporters

are

highly

specifically

stage

and

tissue-regulated

and

transport

special

substrates

such

as

neurotransmitters

-LRB-

ACC

-RRB-

in

neural

cells

or

carbohydrates

-LRB-

POT

-RRB-

in

leaf

tissue

Comparison

of

the

overall

gene

compositions

of

bacteria

-LRB-

E.

coli

-RRB-

animals

-LRB-

H.

sapiens

-RRB-

and

plants

-LRB-

A.

thaliana

and

O.

sativa

-RRB-

reveals

the

strategies

for

osmotic

pressure

adjustment

and

the

features

of

the

substance-transport

systems

in

each

organism

-LRB-

Fig.

-RRB-

Since

a

monad

can

not

control

the

ion

environment

of

its

external

world

and

has

no

need

to

communicate

with

other

cells

the

role

of

its

transporter

proteins

is

restricted

to

the

control

of

material

transport

into

and

out

of

the

cell

Therefore

transport

depends

mainly

on

an

energy-consuming

system

-LRB-

pump

-RRB-

and

an

internal

ion-gradient-dependent

system

-LRB-

secondary

transporter

-RRB-

Because

the

bacterium

has

a

cell

wall

osmotic

pressure

is

opposed

by

cell

wall

pressure

and

there

is

no

need

to

form

a

molecular

concentration

gradient

to

prevent

excessive

accumulation

within

the

cell

Additionally

bacteria

are

small

-LRB-

μm

of

the

size

of

an

animal

cell

-RRB-

and

do

not

have

many

of

the

organelles

and

membrane

structures

possessed

by

higher

organisms

Therefore

transport

of

substances

is

simple

and

it

is

easy

to

control

their

concentrations

in

the

cell

The

genes

encoding

the

ATP-dependent

pump

-LRB-

ABC

-RRB-

phosphotransferase

-LRB-

PTS

-RRB-

small

conductance

mechanosensitive

ion

channel

-LRB-

MscS

-RRB-

and

secondary

active

transporters

-LRB-

e.g.

APC

DMT

MFS

RND

-RRB-

with

H

or

as

co-transporter

molecules

have

diverged

in

bacteria

-LRB-

Fig.

-RRB-

Fig.

of

comparison

of

membrane

transport

genes

in

bacteria

animals

and

plants

Bacteria-specific

genes

blue

animal-specific

genes

red

plant-specific

genes

green

bacteria

and

animal-specific

genes

purple

bacteria

and

plant-specific

genes

brown

genes

with

divergent

numbers

in

organisms

yellow

ABC

ATP-binding

Cassette

ArsAB

Arsenite-Antimonite

Efflux

F-ATPase

H

or

translocating

F-type

V-type

and

A-type

ATPase

H

Ppase

H

translocating

Pyrophosphatase

IISP

General

Secretory

Pathway

-LRB-

Sec

-RRB-

MPT

Mitochondrial

Protein

Translocase

P-ATPase

P-type

ATPase

ACC

ATP-gated

Cation

Channel

Annexin

Annexin

Bestrophin

Anion

Channel-forming

Bestrophin

Channel

ClC

Chloride

Channel

Connexin

Gap

Junction-forming

Connexin

CSC

Chloroplast

Outer

Envelope

Solute

Channel

CytB

Phagocyte

NADPH

Oxidase-associated

Cytochrome

-LRB-

CytB

-RRB-

H

channel

E-ClC

Epithelial

Chloride

Channel

EnaC

Epithelial

Channel

GIC

Glutamate-gated

Ion

Channel

Cation

Channel-forming

Heat

Shock

ICC

Intracellular

Chloride

Channel

Ic

ln

Nucleotide-sensitive

Anion-selective

Channel

Innexin

Gap

Junction-forming

Innexin

IRK-C

Inward

Rectifier

K

Channel

LIC

Ligand-gated

Ion

Channel

of

Neurotransmitter

Receptors

Yeast

Stretch-Activated

Cation-Selective

Channel

MIP

Major

Intrinsic

Protein

MIT

CorA

Metal

Ion

Transporter

MscL

Large

Conductance

Mechanosensitive

Ion

Channel

MscS

Small

Conductance

Mechanosensitive

Ion

Channel

Non-selective

Cation

O-ClC

Organellar

Chloride

Channel

PCC

Polycystin

Cation

Channel

PLM

Phospholemman

RIR-CaC

Ryanodine-Inositol

Receptor

Channel

Chloroplast

Envelope

Anion

Channel-forming

TRP-CC

Transient

Receptor

Potential

Channel

UT

Urea

Transporter

VIC

Voltage-gated

Ion

Channel

AAA

ATP

ADP

Antiporter

AAAP

Amino

Acid/Auxin

Permease

AAE

Aspartate

Alanine

Exchanger

AbgT

p-Aminobenzoyl-glutamate

Transporter

Arsenical

AE

Anion

Exchanger

AEC

Auxin

Efflux

Carrier

AGCS

Alanine

or

Glycine

Cation

Symporter

Amt

Ammonium

or

Ammonia

Transporter

APC

Amino

Acid-Polyamine-Organocation

ArsB

Arsenite-Antimonite

-LRB-

ArsB

-RRB-

Efflux

BASS

Bile

Acid

Symporter

BCCT

Betaine/Carnitine/Choline

Transporter

BenE

Benzoate

H

Symporter

CaCA

Cation

Antiporter

CCC

Cation-Chloride

Cotransporter

CDF

Cation

Diffusion

Facilitator

CHR

Chromate

Ion

Transporter

CNT

Concentrative

Nucleoside

Transporter

Monovalent

Cation

Proton

Monovalent

Cation

Proton

DAACS

Dicarboxylate/Amino

Acid

Cation

-LRB-

or

H

-RRB-

Symporter

DASS

Divalent

Anion

Symporter

Dcu

Uptake

DcuC

Uptake

C

DMT

Drug/Metabolite

Transporter

ENT

Equilibrative

Nucleoside

Transporter

ESS

Glutamate

Symporter

FBT

Folate-Biopterin

Transporter

FNT

Formate-Nitrite

Transporter

GntP

Gluconate

H

Symporter

GPH

Glycoside-Pentoside-Hexuronide

-LRB-

GPH

-RRB-

Cation

Symporter

GUP

Glycerol

Uptake

HAAAP

Hydroxy/Aromatic

Amino

Acid

Permease

KDGT

Transporter

KUP

K

Uptake

Permease

LCT

Lysosomal

Cystine

Transporter

LctP

Lactate

Permease

LIV-E

Branched

Chain

Amino

Acid

Exporter

LIVCS

Branched

Chain

Amino

Acid

Cation

Symporter

LysE

L-Lysine

Exporter

MC

Mitochondrial

Carrier

MET

TMS

Multidrug

Endosomal

Transporter

MFS

Major

Facilitator

Superfamily

MOP

Flippase

Superfamily

MTC

Mitochondrial

Tricarboxylate

Carrier

Nucleobase

Cation

Nucleobase

Cation

NhaA

H

Antiporter

A

NhaB

H

Antiporter

B

NhaD

H

Antiporter

D

NiCoT

Transporter

Nramp

Metal

Ion

-LRB-

iron

-RRB-

Transporter

NSS

Neurotransmitter

Sodium

Symporter

OAT

Organo

Anion

Transporter

OPT

Oligopeptide

Transporter

OST

Organic

Solute

Transporter

Cytochrome

Oxidase

Biogenesis

PiT

Inorganic

Phosphate

Transporter

PnaS

Phosphate

Symporter

POT

Proton-dependent

Oligopeptide

Transporter

RFC

Reduced

Folate

Carrier

RhtB

Resistance

to

Homoserine/Threonine

RND

Resistance-Nodulation-Cell

Division

SSS

Solute

Sodium

Symporter

SulP

Sulfate

Permease

Tat

Twin

Arginine

Targeting

TDT

Tellurite-resistance/Dicarboxylate

Transporter

ThrE

Threonine/Serine

Exporter

TRAP-T

Tripartite

ATP-independent

Periplasmic

Transporter

Trk

K

Transporter

ZIP

Zinc

-LRB-

-RRB-

Iron

-LRB-

-RRB-

Permease

GPTS

general

carbohydrate

phosphotransferase

SSPTS

sugar

specific

phosphotransferase

Dipicolinic

Acid

Transporter

Copper

Transporter

FeoB

Ferrous

Iron

Uptake

FeT

Low

Affinity

Transporter

FP

Ferroportin

LPI

Lysosomal

Protein

Import

OfeT

Iron/Lead

Transporter

PnuC

nicotinamide

mononucleotide

-LRB-

NMN

-RRB-

uptake

permease

PPI

Peroxisomal

Protein

Importer

PUP

Peptide

Uptake

or

Activated

Fatty

Acid

Export

Permease

Increases

in

the

size

and

complexity

of

cells

with

the

emergence

of

organelles

and

an

internal

membrane

structure

arose

in

the

process

of

evolution

from

prokaryote

to

eukaryote

The

eukaryote

's

inclusion

of

the

prokaryote

's

transport

genes

in

the

mitochondrion

or

chloroplast

allowed

the

transport

of

homeostatic

substrate

to

organelles

as

well

as

other

materials

and

the

products

of

specific

biosynthesis

-LRB-

tricarboxylic

acid

cycle

electron-transport

chain

and

photosynthesis

-RRB-

Some

of

the

genes

are

indistinguishable

between

eukaryotic

transcript

and

organelle

transporter

but

there

are

typical

organelle

transporters

-LRB-

e.g.

MC

MCX

Tat

-RRB-

in

eukaryotes

and

they

make

a

large

group

-LRB-

of

all

transporters

-RRB-

Furthermore

with

the

development

of

multicellular

eukaryotes

there

came

a

need

for

organisms

to

develop

a

system

of

transmission

between

cells

Animals

and

plants

developed

specific

systems

to

solve

the

problem

Animals

lost

the

cell

wall

structure

and

gained

cellular

mobility

and

flexibility

which

also

promoted

integration

of

the

whole

body

and

specialization

of

tissues

and

cells

The

loss

of

the

cell

wall

also

led

to

the

need

to

control

osmotic

pressure

against

the

plasma

membrane

Animal

cells

regulate

osmotic

pressure

by

raising

the

concentration

outside

the

plasma

membrane

by

active

transport

with

K

ATPase

This

gradient

is

used

not

only

for

homeostasis

of

the

osmotic

pressure

of

the

membrane

but

also

as

an

electrical

potential

to

control

the

voltage-dependent

channel

system

and

for

co-transport

of

symport

or

antiport

molecules

of

secondary

transportation

Animals

then

developed

the

channel

system

-LRB-

e.g.

connexin

E-ClC

EnaC

ICC

Ic

ln

LIC

PCC

PLM

RIR-CaC

VIC

-RRB-

for

rapid

transmission

in

nerve

and

muscle

tissues

together

with

an

gradient

dependent

on

the

secondary

transport

system

-LRB-

e.g.

NSS

PnaS

SSS

-RRB-

This

ion

gradient

adaptation

system

is

a

feature

of

the

newly

added

transporters

and

has

evolved

with

animal

specificities

i.e.

the

need

for

rapid

movement

and

adaptation

to

environmental

stimulation

In

contrast

to

animals

plants

have

retained

the

cell

wall

instead

of

acquiring

mobility

Because

their

cell

structures

and

sizes

became

complicated

and

large

plants

also

evolved

a

means

of

controlling

osmotic

pressure

this

means

differed

from

the

cell-wall-dependent

system

of

bacteria

Because

the

plant

cell

controls

osmotic

pressure

by

the

turgor

pressure

of

the

cell

wall

it

does

not

require

a

sodium

or

other

ion

gradient

and

lacks

an

K

ATPase

for

control

of

osmotic

pressure

against

the

plasma

membrane

On

the

other

hand

an

increase

in

permeability

to

water

is

necessary

for

smooth

transmission

of

water

pressure

in

the

large

and

complicated

structures

of

the

cell

Therefore

water

channels

are

abundant

in

both

the

plasma

membrane

and

the

vacuoles

-LRB-

of

membrane

proteins

-RRB-

and

the

diversity

of

water

channels

-LRB-

MIP

-RRB-

is

also

four

to

five

times

higher

than

that

in

bacteria

and

animals

Compared

with

circulation

systems

in

animals

whole-plant

circulation

systems

are

simple

and

less

controlled

Plant

cells

also

need

to

be

more

self-complementary

in

their

homeostasis

Additionally

plants

have

less

choice

in

their

environmental

conditions

-LRB-

e.g.

soil

weather

stress

-RRB-

and

must

have

more

transport

systems

for

cell

homeostasis

than

animals

This

is

why

plant

cells

include

vacuoles

to

pool

many

substances

and

bioactive

compounds

Because

plants

obtain

energy

through

photosynthesis

there

are

more

ATP-dependent

-LRB-

pump

-RRB-

transporters

-LRB-

ABC

P-type

ATPase

-RRB-

in

their

cells

than

in

animal

cells

Unlike

the

case

in

animals

voltage-dependent

systems

and

ion-dependent

secondary

transport

systems

have

not

developed

in

plants

systems

that

rely

on

H

rather

than

have

developed

as

part

of

plant

secondary

transporter

systems

Because

the

increase

in

H

brings

acidification

plants

maintain

the

neutrality

of

the

cytoplasm

by

accumulating

H

in

vacuoles

which

are

membrane-bound

organelles

separated

from

the

other

cytoplasmic

contents

By

having

an

internal

ion

pool

plant

cells

can

control

the

ion

gradient

easily

and

safely

Therefore

secondary

transport

genes

-LRB-

e.g.

DMT

MFS

MOP

POT

-RRB-

that

use

H

as

a

co-transporter

molecule

have

diverged

far

in

plants

-LRB-

Fig.

-RRB-

The

inclusion

of

pooling

and

energy-synthesizing

organelles

in

the

cell

may

be

why

plant

cells

-LRB-

μm

-RRB-

are

larger

than

animal

cells

-LRB-

μm

-RRB-

Conclusions

Comparative

analysis

of

membrane

transporters

among

these

eight

diverse

organisms

indicates

the

type

of

cell

homeostasis

as

evidenced

by

the

pattern

of

gene

conservation

and

diversification

Evolutionary

changes

in

gene

families

in

general

indicate

the

dynamics

of

alterations

in

biological

systems

and

gene

networks

Therefore

analysis

of

large

categories

of

gene

families

may

reveal

many

basic

concepts

of

biological

systems

In

practice

analyses

of

the

membrane

transporter

mechanism

are

useful

in

revealing

changes

in

the

absorption

of

molecules

by

or

their

efflux

from

cells

and

tissues

This

information

also

is

useful

for

examining

changes

in

soil

adaptability

nutritional

demand

and

stress

tolerance

in

plants

It

may

also

help

to

improve

the

harvest

of

crop

cultivars

or

extend

areas

habitable

by

plant

species

Gene

networks

are

intricately

related

and

analysis

of

the

whole

genetic

structure

is

needed

to

gain

a

full

understanding

of

biological

phenomena

and

systems

of

gene

regulation

We

are

continuing

to

analyze

whole

categories

of

genes

in

an

effort

to

develop

an

overview

of

total

gene

networks

Electronic

supplementary

material

-LRB-

DOC

kb

-RRB-

-LRB-

XLS

kb

-RRB-

-LRB-

PDF

kb

-RRB-

-LRB-

PDF

kb

-RRB-

-LRB-

PDF

kb

-RRB-