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10
Chapter 1
Why study membrane transport by LacS
Cells of all living creatures are able to maintain their specific chemical
composition by shielding their interior from the exterior with a largely
impermeable membrane, the lipid bilayer. The same holds true for the membrane
bound compartments in eukaryotic cells like the endoplasmatic reticulum, the
GOLGI, peroxi-, lyso-, or endosomes, mitochondria, chloroplasts and to some
extent the nucleus. In the transmission of information and solutes across the
biological membrane integral membrane proteins play a central role. The
importance of these proteins is reflected in the genomes sequenced thus far. Based
on experimental and bio-informatical data, including topology and domain
predictions and sequence alignments, about 30% of the gene products are believed
to be polytopic transmembrane proteins (149). By a specialized class of integral
membrane transport proteins, solutes can move across the hydrophobic barrier of
the phospholipid bilayer. The communicating pathway provided by these specific
membrane spanning proteins allows for the uptake of nutrients, ions and
metabolites, and communication between cells and the surrounding environment.
A few examples of important transport reaction in humans are the disposal of CO2
from red blood cells by the HCO3-/Cl- exchanger (14), the reabsorbtion of glucose
in the kidneys by the renal glucose transporters (157) and the removal of
neurotransmitter in the synaptic endings of neurons by the e.g. the glutamate
transporters (39).
With transport being one of the key processes in life, understanding the
transducers of this reaction represents an important challenge in biology. The
interest in the structure and function of the transport protein that is central in this
thesis, the lactose transport protein (LacS) from Streptococcus thermophilus, is
thus of fundamental nature. Understanding this particular transport protein in
terms of structure -function relationships and dynamics hopefully helps to
formulate rules that are more generally applicable. In addition, methodology
developed for the study of this protein could be of more general use.
Classes of membrane transport proteins
Transport proteins are classified in four groups on the basis of their
energy coupling mechanism (125) (www-bilogy.ucsd.edu/~msaier/transport/
titlepage.html). The channels and pores allow passive diffusion of specific solutes
through their aqueous interior resulting in an equal distribution of solutes over the
membrane (Fig. 1D). The primary transporters energize transport by use of
primary energy sources such as light or energy from chemical conversions. A
large number of these belong to the ATP-binding cassette (ABC)-superfamily that
energize transport via hydrolysis of ATP (58) (Fig. 1A). The secondary
transporters use the free energy that is stored in electrochemical gradients of
solutes over the membrane to drive uniport, symport or antiport (108) (Fig. 1B).
By far the largest family of secondary transporters is the Major Facilitator
Superfamily (MFS), comprising over 1000 members from all three kingdoms of
Introduction
11
life (102). A fourth group of transporters concerns the group translocation systems
that couple translocation to a chemical modification of the substrate. The
phosphoenolpyruvate dependent phosphotransferase (PTS) systems phosphorylate
the substrate concomitant with transport and are found only in bacteria (119) (Fig.
1C). Carbohydrate transport across the membrane of eukaryotes is dominated by
the activities of permeases that fall within the MFS, whereas in prokaryotes they
are also transported by members of the ABC and PTS superfamilies (126).
The secondary transporters are generally composed of a single
polypeptide folded into several transmembrane spanning α-helical segments,
connected by intracellular and extracellular loops. In addition to a transmembrane
carrier domain composed of α-helical structural elements, ABC transporters have
domains responsible for ATP hydrolysis, and those that function in the uptake of
solutes into the cell employ a specific ligand binding protein or domain to capture
the solute. The separation into these groups of transporters with each distinct
structural and functional features is not absolute. Transport systems with
functional features of both secondary and ABC transporters have been found, e.g.
secondary transport systems that make use of a substrate binding protein, or
secondary transporters that convert into primary systems upon association with an
ATPase (25). Overlap with the structural features characteristic of channels is
found in secondary glutamate transporters, where reentrant loop structures are
proposed to exist, and possibly reflect the channel-like properties associated with
some of these transporters (135).
Figure 1:Examples of energy coupling mechanisms and structural organization of different
transport proteins. A) Primary transporters like those from the ABC-superfamily, B) secondary
transport proteins like those from the MF-superfamily, C) group translocators like those from the
PTS-superfamily, and D) passive diffusion through channels and pores.
12
Chapter 1
The GPH-family of Major Facilitators
The Major Facilitator Superfamily. The topology of proteins from the
MFS typically comprise of 12 transmembrane spanning α-helices of which the
first six exhibit sequence similarity with the last. It is believed that these proteins
arose by internal tandem gene duplication events. Three of the established 28
families of the MFS have 14 instead of 12 transmembrane spanning α-helices
(102). Functionally the transporters from the MFS are very diverse. A wide range
of substrates is transported, among which sugars, polyols, drugs,
neurotransmitters, Krebs cycle metabolites, phosphorylated glycolytic
intermediates, amino acids, peptides, compatible solutes, nucleosides and
(in)organic ions. In very few cases MFS members function as receptors and
modulate gene expression (63, 76). The transport mechanisms vary as well, and
MFS members catalyze import and export reactions, or allow mere equilibration
of substrates over the membrane. Equilibration of a neutral substrate is catalyzed
by uniport systems, whereas solute accumulation usually requires that transport is
coupled to the co-transport (symport) of monovalent cations, mostly H+ or Na+.
When a substrate is pumped out against its concentration gradient, energy
coupling involves substrate:H+ or Na+ countertransport (antiport). Some carriers
preferentially or obligatorily catalyze solute:solute antiport, where two pools of
solutes move in opposite directions over the membrane (exchange). Many
secondary carriers can catalyze transport in both directions depending on the
direction of the electrochemical proton or sodium gradient.
The Galactoside-Pentoside-Hexuronide(GPH):cation symporter family.
The members of the GPH-family facilitate the uptake of Galactosides, Pentosides
and Hexuronides and use Na+, H+ and/or Li+ as coupling ion (107). Although the
GPH members show only marginal sequence similarity with other members of the
MFS, motif/matrix analysis clearly suggests that these proteins arose from a
common ancestor, making it almost certain a distant constituent of the MFS (126).
A phylogenetic analysis of sugar transporters from bacteria, yeast, humans and
plants is shown in figure 2 (taken from (118). Most sequenced transporters of the
GPH-family are from bacteria but eukaryotic members have been identified, e.g.,
the sucrose transporters from plants and fungi (94, 118). Sequence comparison
indicates three GPH-subfamilies among the bacterial members, namely the MelB,
LacS and GusB subfamilies with as little as approximately 25% identity between
the subfamilies. Both the amphipathic nature of helix II and the high sequence
conservation in interhelix loop 10-11 and helix XI are features shared by these
proteins. Based on bio-informatical information, a putative transporter from
Caenorhabditis elegans is predicted to belong to the GPH family and shares these
characteristics (www.biology.ucsd.edu/~ipaulsen/transport/). Some members, like
the LacS proteins from S. thermophilus and Lactobacillus bulgaricus have a
carboxyl-terminal cytoplasmic domain of 160 amino acids, named IIA because of
its similarity to IIA domains of various PTS systems (110).
Introduction
13
Figure 2: Phylogenetic analysis of secondary sugar transporters from bacteria, yeast, humans and
plants (taken from (118)). Bootstrap analysis was performed on aligned amino acid sequences of the
lactose transporter (LacY), the melibiose transporter (MelB) and the glucuronide transporter (GusB)
from Escherichia coli, the pentoside transporter (YnaJ) from Bacillus subtilis, the isoprimeverose
transporter (XylP) from Lactobacillus pentosus, the raffinose transporter (RafP) from Pediococcus
pentosaceus, the lactose transporter from S. thermophilus, the maltose transporter (Mal11), the αglucoside transporter (AGT1) and the hexose carrier (HXT1) from Saccharomyces cerevisiae, the αglucoside transporter (pSUT1) and glucose transporter (GHT1) from Schizosaccharomyces pombe,
the monosaccharide transporter (STP1) and sucrose transporters (SUC2, SUT2, SUT4) from
Arabidopsis thaliana, the sucrose transporters (SUT1) from Zea mays and Solanum tuberosum and
the human Na+/glucose cotransporter (SGLT1). The GPH members are underlined, LacY is a
member of the oligosaccharide:H+ symporter (OHS) family and the others belong to the Sugar Porter
families.
The lactose transport protein of Streptococcus thermophilus
The lactose transport protein (LacS) of the lactic
Biological function.
acid bacterium Streptococcus thermophilus is functional in the cytoplasmic
membrane and responsible for the uptake of sugars from the medium. S.
thermophilus can grow on lactose as sole energy source. When growing in milk
relatively high expression levels are obtained and the protein catalyzes
stoichiometric exchange of lactose and galactose, the product of internal
hydrolysis of lactose by β-galactosidase (73, 104). Although the genes for
galactose metabolism are present on the chromosome, the complete metabolic
pathway is not expressed and consequently galactose is excreted into the medium
and only the glucose moiety of lactose is metabolized.
Transport mechanism. Membrane transport proteins like LacS are
proposed to catalyze transport by an 'alternating access' mode of transport, that is,
they alternate between at minimum two fundamentally different conformations in
which the binding site is accessible from either side of the membrane, the outside
facing (Eout) and the inside facing conformations (Ein) (Fig. 3).
14
Chapter 1
Figure 3: Two binding conformers of LacS, one exposing the binding site to the extracellular
surface (Eout, left) and one where the binding site is accessible from the inside (Ein, right). The
apparent affinity constants for lactose are approximately 5 mM and 0.2 mM at the outside and inside
facing binding site, respectively. These constants are derived from transport kinetics rather than
equilibrium binding.
A priori, one would expect that sugars bind with a higher affinity to the
outside facing binding site than to the inside facing binding site, that is the site
from where the sugar, which is taken up, has to be released. However, this is not
necessarily true for a system like LacS, where following release of lactose,
galactose is bound to the inside facing binding site, and, after reorientation of the
binding site, released from the outside facing binding site. Indeed, the LacS
protein displays a pronounced asymmetry in apparent binding affinities for lactose
at the inside and outside facing binding sites, the apparent affinity constant at the
inside being about one order of magnitude smaller (73).
For a coupled transport of proton (H) and sugar (S), the switch from the
outside facing to the inside facing conformation should only occur when both
substrates are bound, that is, when a ternary complex of Enzyme-Sugar-H+ (ESH)
is formed (77). Mutants in which the conversions from inside facing to outside
facing and vice versa do occur with either of the two ligands bound, EH or ES,
induce so-called EH or ES leak pathways, and facilitate proton or solute uniport in
stead of catalyzing a symport reaction (82). The most simple reaction schemes for
influx, ∆p-driven uptake, exchange and efflux are depicted in figure 4. The
conformations via which transport occurs are presumably identical in the different
transport cycles, implying that also in influx, efflux and exchange protons are part
of the transport cycle (105). However, the steps and the order/direction in which
they are performed are different, and the concentrations of substrate and proton
and the kinetics of association of these ligands with the carrier protein determine
which of the translocation cycles is operative. The conformational changes are
slower when the binding sites are empty, as the catalysis of exchange, in which
such a conformational change does not occur, is about an order of magnitude
faster than the fastest transport involving isomerization of empty binding sites
(73). Therefore, in the presence of substrates at both sides of the membrane
15
Introduction
exchange is dominant, whereas efflux or influx occur when sugars are present at
either the outside or inside of the membrane, respectively. These reactions are
driven by the concentration gradients of the sugars across the membrane (33).
Accumulation of a sugar occurs in the presence of a proton motive force via the
same reaction cycle as depicted for influx and is driven by the electrochemical
gradient of protons across the membrane. Apparent accumulation of external
sugar can also take place via the exchange mode of transport, provided the
concentration gradient of a second sugar is in the opposite direction.
ESH
ESH
in
E in
E
in
in
in
ESH
out
ESH
E
out
A
E
in
ESH
out
ESH
out
ESH
in
out
out
ESH
out
E
E
B
C
Figure 4: Simplified reaction cycles of influx and ∆p-driven uptake (a), exchange (b) and efflux
(c) in an alternating access mode of transport. 'Ein' and 'Eout' represent conformations of the carrier
in which a binding site is accessible from the inside and outside of the cell, respectively. 'S'
represents the sugar substrate and 'H' the proton.
Regulation of transport activity. The work of Marga Gunnewijk has
shown how metabolism and transport are well tuned to the carbohydrate
availability and the metabolic capacity of the cell (45, 46, 47, 47a). The regulation
is such that upon decreased glycolytic activity, e.g., due to a lowered availability
of lactose in the medium, the transport capacity is increased by an upregulation of
both the expression and the transport activity of LacS. One of the signaling routes
senses the ATP and free Pi levels in the cell and regulates the transcription of the
lacS gene via HPr(Ser-P)/CcpA-mediated repression. The other route senses the
PEP/pyruvate levels and regulates the activity of the LacS carrier via HPr(His~P)
mediated phosphorylation of the IIA domain of LacS. Phosphorylation differently
affects ∆p-driven transport and exchange transport by specifically increasing the
Vmax of exchange transport, and not of ∆p-driven lactose uptake. How the
phosphorylation state of the IIA domain is communicated to the carrier domain
and alters the transport kinetics is not understood to date.
Structure.
Like most other members of the MFS, LacS is predicted
to consist of 12 transmembrane spanning α-helical segments that are connected by
intra- and extracellular loops, but, in addition, the protein has a carboxyl terminal
cytoplasmic IIA domain (Fig. 5). High resolution 3D-structures from other
integral membrane proteins with α-helical structural elements show that the α-
16
Chapter 1
helices are tightly packed in the membrane, and often the polar faces of α-helices
are buried in the interior of the protein, whereas the more a-polar faces are
oriented towards the lipids (27, 116, 117). The α-helices are oriented more or less
perpendicular to the membrane plane and are generally composed of
approximately 20 amino acids with largely hydrophobic side chains. High
resolution structural data is so far absent for secondary transport proteins, but the
medium or low resolution structures that are available from 2D and 3D crystals
have revealed information on the size and shape of the proteins and the packing of
α-helices (141, 153, 154, 159, 163). In the absence of high-resolution 3D
structures, information on the structure of LacS has been obtained by a variety of
strategies, of which some will be discussed in the coming chapters. One of the
approaches that we have taken, but which is not described further in this thesis,
involves solid state NMR using sequence selective labeling of the LacS protein.
The distance information obtained from the interaction between [1-13C]Dgalactose bound to LacS in native membranes and a nitroxide label attached to a
cysteine in interhelix loop 10-11 of LacS, together with the low mobility of this
label, suggested that the inter-helical loop 10-11 in LacS is folded into the
membrane and likely located between transmembrane spanning α-helices
involved in substrate binding (137).
Figure 5: The fold of the LacS protein, predicted from hydropathy and reporter fusion analysis with
12 transmembrane spanning α-helices and the IIA domain.
Introduction
17
Experimental tools.
Most
biochemical
and
biophysical
characterizations require that the protein of interest is in a purified form and that
ligand binding or transport can be measured. The work of Jan Knol has provided
an elegant, reproducible method for the overexpression, purification and
reconstitution of LacS into artificial lipid bilayers, yielding so-called
proteoliposomes in which the LacS protein is functionally incorporated in the lipid
bilayer (72, 73). The development of this methodology has been crucial for many
of the studies described in this thesis. One of the essential features is the fact that
reconstitution proceeds unidirectionally, that is, the majority of LacS is inserted in
the lipid bilayer with the IIA domain at the outside of the proteoliposomes, which
is in-side-out compared to the in vivo situation.
For reconstitution, liposomes are saturated with Triton-X-100 in order to
destabilize the lipid bilayer so that insertion of proteins is enabled. It was
proposed that reconstitution is uni-directional as Triton X-100 doped liposomes
stay spherical and intact, and membrane proteins like LacS that have a large
domain will insert such that the membrane domain enters the lipid/detergent
bilayer while the soluble domain rests in the aqueous environment. When these
proteoliposomes are quickly frozen in liquid nitrogen and slowly thawed, the lipid
bilayers fuse and large multi-lamellar structures are obtained. After extrusion
through a 200 nm filter uni-lamellar approximately equally sized structures are
obtained. The contents of the proteoliposomes can be modulated upon addition of
the required solutes, buffer components, or proteins during the freeze-thawextrusion procedure (26).
Outline of the thesis
In order to understand how transport is accomplished in a secondary
transport system we need information on function, structure and dynamics.
Provided that one understands the relations between these parameters, knowledge
about the function will give information about the structure and vice versa. The
approaches described in chapters 2, 3 and 4 rely on functional assays, from which
we aim to deduce information about the function as well as the structure of LacS.
In chapter 2, we measure binding and transport of a range of sugars in both
transport directions from which we deduce architectural details of the outside and
inside facing binding sites and the translocation pathway. In the third chapter, the
functional analyses of site-directed and second-site suppressor mutants were used
to identify areas and residues in the protein involved in proton and/or sugar
binding. Based on these and previously published data a model is proposed with
the catalyticly important regions lining the binding site(s) and translocation
pathway. In the chapters 4, 5 and 6, the quaternary structure and function of LacS
and other secondary transport proteins is described. Analytical ultra-centrifugation
and freeze fracture EM revealed that LacS and its homologue XylP form dimers in
the membrane as well as in detergent solution (37) (chapter 5). For LacS, this
structural phenomenon is essential for the catalysis of ∆p-driven uptake but not for
18
Chapter 1
the catalysis of exchange, as is shown in chapter 4. The use of Blue Native
electrophoresis for quaternary structure determination is evaluated in chapter 5
using LacS and XylP as test-cases. The current knowledge on quaternary structure
and function of secondary transport proteins in general, and the techniques used
for these kinds of studies are discussed in chapter 6. In chapter 7 we reflect on the
approaches taken and combine all functional and structural data to describe the
present level of understanding the transport catalyzed by LacS. We evaluate to
what extent the insights could be more generally true for secondary transport
proteins.