Secondary active transport
use the ion gradients established by ATPase for
transport of various substances against their
gradients of electrochemical potentials via
transporters/ carriers.
Transporters tend to be highly specific for their substrates,
therefore the cell has many diverse transporters in the
membrane.
1
The carriers/porters form complexes by binding their solutes
before transporting them across the bilayer by the secondary
processes of symport or antiport. They are also called electrical
potential-driven transporters.
Class of carriers
 a very large family is called the major facilitator
superfamily (MFS).
 ionophores, nonribosomally synthesized carriers (e.g.
valinomycin and nigericin)
 Ton B, family of proteins involved in transferring energy to
the outer membrane of Gram-negative bacteria.
Luckey, M. 2008. Membrane Structural Biology.
2
Major Facilitator Superfamily (MFS)
Lactose permease (LacY) and the glycerol-3-phosphate transporter (GlpT)
of E. coli are members of the MFS of secondary active transporters.
LacY, a galactoside/H+ symporter, utilizes the proton gradients of the inner
membrane to drive the 100-fold accumulation of lactose inside cell.
GlpT, an organophosphate/phosphate antiporter, takes up glycerol-3phosphate for use as an energy source and as a precursor for
phospholipids.
X-ray structure of LacY
Luckey, M. 2008. Membrane Structural Biology.
3
GlpT. Model of conformational changes proposed to accompany substrate
translocation (rocker-switch transporters)
The protein alternates between an
outward-facing conformation (Co) and
inward-facing conformation (Ci). The
substrate phosphate (red) is shown as
it moves from the inside (C) to the
outside (A). The crystal structure
corresponds to the inward-facing
conformation without substrate (D).
The outward-facing conformation was
generated by rotating the two halves of
GlpT by 16° each in opposite direction
(B), while the Co-Pi conformation (A)
required a 10° rotation.
Luckey, M. 2008. Membrane Structural Biology.
The common architecture, and likely mechanism, of MFS transporters does
not entirely reveal how these uniporters, symporters, and antiporters for
widely varied substrates carry out secondary active transport.
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Leucine transporter
Members of the solute carrier 6 (SLC6) family of sodium-coupled
transporters, also known as neurotransmitter sodium symporters,
make up one of the most widely investigated and pharmacologically important classes.
SLC6 proteins play a central role in diverse physiological
processes, ranging from the maintenance of cellular osmotic
pressure to the reuptake of small-molecule neurotransmitters in
the brain. SLC6 dysfunction is implicated in numerous debilitating
illnesses, such as depression (8), obsessive-compulsive disorder,
epilepsy, autism, orthostatic intolerance, X-linked creatinedeficiency syndrome, and retinal degeneration. The transport
activity of these molecular machines can be inhibited by many
different compounds, including tricyclic antidepressants (TCAs),
selective serotonergic reuptake inhibitors, anticonvulsants, and
cocaine.
LeuT occluded-state structures.
Superposition of the LeuT-Leu (gray),
LeuT-Ala (green), LeuT-Gly (magenta),
LeuT-Met (blue), and L-4-F-Phe (orange)
complexes by use of α-carbon positions.
Membrane boundaries are demarcated by
the two solid black lines.
Singh et al.. 2008. Sci. 322:1655.
Unraveling the molecular principles that define a substrate (a
molecule that can be transported) versus a competitive inhibitor
(a molecule that can displace the substrate but is not itself
transported) is intimately linked to the larger goal of elucidating
transport mechanism and ultimately to the development of new
therapeutic agents.
The leucine transporter (LeuT), a prokaryotic SLC6 member (18),
provides an opportunity to couple functional and structural data
to uncover the molecular mechanisms of transport and inhibition.
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Amino acid transporters (ApcT)
Water
Amino acid, polyamine, and
organocation (APC) transporters
are members of a large family of
secondary transport proteins that
catalyze the uniport, symport, and
antiport of a broad range of
substrates across the membrane
bilayer.
ApcT is a broad-specificity amino acid transporter.
Architecture of ApcT. (A) Ribbon diagram of the ApcT structure, viewed parallel to the membrane, along the pseudo 2-fold axis of
molecular symmetry. (B) "Top" down view of ApcT from the outside. (C) Slice through a solvent-accessible surface of ApcT showing a
solvent-accessible pathway reaching deep into the transporter. Water molecules are shown as cyan spheres. (D) Superposition of the
scaffold helices TMs 3 to 5 and 8 to 10 of ApcT and vSGLT onto the equivalent elements of LeuT shows that in ApcT, TM1b is closed to
the outside and TM1a is partially open to the inside. TMs 2 to 10 of LeuT are shown as an α-carbon trace.
Shaffer. 2009. Sci. 325:1010.
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Mitochondrial ADP/ATP Carrier
Mitochondria are surrounded by two membranes: an outer membrane with pores
that allow fairly nonselective passage of molecules and ions up to around 500 Da,
and an inner membrane with at least 20 specific transport functions.
The ADP/ATP carrier (AAC) provides the ADP
substrate needed inside the matrix for ATP
synthase and to export the ATP it produces.
The AAC is an electrogenic antiporter that
exchange one ADP3- for one ATP4- without
bound Mg2+ ions, with the net export of one
negative charge driven by the membrane
potential.
Indispensable to the generation of ATP by
respiration, AAC is the most abundant of the
mitochondrial carriers and makes up 10% of
the protein extracted from the inner
mitochondrial membrane.
8
Facilitated diffusion through ion transporters
Ion transporters are differentiated from channels by their much
slower rate of turnover, by thermodynamic differences
(transporters have one infinite energy barrier in contrast to finite
barriers at all times in channels) and by their primary structure.
9
Transporters/Carriers exhibit saturation kinetics (Vmax).
Permeation of ions involves binding and de-binding of
ions to a specific site, together with conformation
changes of the proteins.
(A)
(B)
Km is Michaelis-Menten constant
10
Turnover rates
• Pump
long-range and complex conformation
transitions
• Transporter/ Carrier
conformation changes, but no long-range
interaction with soluble substrates
• Channel
no conformation changes during transport.
Channels have the greatest turnover rate of
all enzymes.
11
Buchanan et al. 2000.
Turnover rates
Transport
system
Molecule transported
per second
2
Pumps
10
Transporters
2
10 -10
5
6
8
Channels
10 -10
12
Buchanan et al. 2000.
Transport proteins in plant plasma membrane
Pumps
Channels
Transporters
13
Overview of the
various transport
processes on the
plasma membrane
and the tonoplast of
plant cells
Cytosol
pH = 7.2
∆E = - 120 mV
= Ecytosol- Eextracellular
Vacuole
pH = 5.5
∆E = - 90 mV
= Evacuole- Eextracellular
14
Taiz and Zeiger. 2006.
Overview of the various transport processes on the tonoplast
Electrical potential, mV
pH∼5.5
pH 7.0-7.4
การปั๊ ม H+ ผ่าน plasma
membrane ออกจาก
เซลล์ ทําให ้ pH ภาย
นอกเซลล์ตํา่ กว่าใน
cytosol
และการปั๊ ม H+ ผ่าน
tonoplast เข ้าสู่
vacuole ทําให ้ pH ของ
vacuole มีคา่ ตํา่ กว่าใน
cytosol
ในทํานองเดียวกัน
ั ย์ไฟฟ้ าของ cytosol
ศก
ตํา่ กว่าภายนอกประมาณ
120 mV และตํา่ กว่า
vacuole 10-30 mV
กล่าวคือ pH ของ
cytosol มีคา่ สูงกว่าอีก
สองบริเวณ และมี
ั ย์ไฟฟ้ าตํา่ สุด
ศก
Cytosol
pH∼5.5
16
Thylakoid (internal) membrane
STROMA (low H+)
LUMEN (high H+)
17
Taiz and Zeiger. 2006.
Mitochondria
Transmembrane transport in plant
mitochondria.
An electrochemical proton gradient
consisting of a membrnae potential
(∆E=-200mV, negative inside) and
a ∆pH (alkaline inside) is established
across the inner mitochondrial
membrane during electron transport.
18
Taiz and Zeiger. 2006.
Mitochondria
19
Taiz and Zeiger. 2006.
Confocal
mapping of
cellular pH via
fluorescence
intensity ratio
imaging.
Rengel. 2002.
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