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1 Amino Acid Transporters: Structure and Molecular Function

1 Amino Acid Transporters: Structure and Molecular Function

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50



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



TABLE 5.1 Amino acid transporters of plasma membranes



AA transporter family



Common acronym



Transport mechanism

designation

(see Fig. 5.1)

AA substrates



Transport

system(s)



SLC1A1-3, A6-7



EAAT1-5



S5



AAA



X2AG



SLC1A4, A5



ASCT1, 2



A2b



SNAA



ASC



SLC6A1, A11, A13



GAT1-3



S4



GABA



GABA



SLC6A5, A9



GLYT1,2



S4



Gly



Gly



SLC6A6



TauT



S4



Tau



Tau



SLC6A7



PROT



S4



Pro



Pro



SLC6A12



BGT1



S4



Betaine, GABA



Human gene

(HGNC)a



SLC1 (2.A.23)

Excitatory AA transporters



SLC6 (2.A.22)

Neurotransmitter transporters



0,1



SLC6A14



ATB



S4



NAA, CAA



B0,1



SLC6A15,

A18, A19



B0AT1-3 (XT2)



S1



NAA



B0



SLC6A17



XT1



S4



NAA



SLC6A20



IMINO (XT3)



S4



Pro, Sarcosine



Pro/β



(i) Cationic AA transporters



SLC7A1-A4



CAT1-4



U



CAA



y1



(ii) Glycoprotein-associated AA

transporters (gpaAT/HAT)c



SLC7A5, A8



LAT1,2



A1



LNAA, TH



L (L1)



SLC7 (2.A.3)



1



SLC7A6, A7



y LAT1,2



A2



NAA, CAA



y1L



SLC7A9



b0,1AT



A1



NAA, CAA,

Cystine



b0,1



SLC7A10



ascT



A1



SNAA



asc



SLC7A11



xCT



A1



Cystine, Glu



X2C



SLC7A13



AGT1



A1



AAA



SLC16A10



TAT1



U



Aromatic AA, TH T



SLC36A1-A4



PAT1-4 (LYAAT)



S2



Pro, Gly, Ala,

GABA



PAT



SLC38A1,A2,A4



SNAT1,2,4



S1



SNAA



A



SLC38A3, A5



SNAT3,5



S3



Gln, Asn, His



N



SLC43A1,A2



LAT3,4



U



LNAA



L (L2)



d



SLC16 (2.A.1)

Monocarboxylate transporters

SLC36 (2.A.18)

Proton-coupled AA transporters

SLC38 (2.A.18)

Small neutral AA transporters



SLC43 (2.A.1)

Large neutral AA transporters

a



Only AA transporters with human orthologues are included.

Na1-dependent NAA antiport, exact mechanism uncertain.

The gpaATs form heteromeric transporters with an “accessory” SLC3 (8.A.9) subunit, either SLC3A1 (rBAT/NBAT) or SLC3A2 (F42hc/CD98).

d

Primarily Na1-NAA/CAA antiport.

First column shows transporter families as classified by Human Genome Organisation (HGNC), see http://www.bioparadigms.org, and (in parentheses) by the

International Union of Biochemistry and Molecular Biology (IUBMB), see http://www.tcdb.org. See also, for example, Broer and Palacin (2011); Hediger et al.

(2013); Schioăth et al. (2013); for original sources and further details on substrate ranges and tissue expression.

AAA, anionic AA; (L/S)NAA, (large/small) neutral AA; CAA, cationic AA; TH, thyroid hormone.

b

c



I. GENERAL AND INTRODUCTORY ASPECTS



51



5.1. AMINO ACID TRANSPORTERS: STRUCTURE AND MOLECULAR FUNCTION



TABLE 5.2



Amino acid transporters of intracellular membranes



Gene family



HGNC



Acronym



AA substrates



Mechanism

(see Fig. 5.1)



Principal localization



SLC17A6-A8



VGLUTs



Glu



A3a



Endosome



SLC25A2, A15



ORNT2, 1



CAA, citrulline



A1b



Mitochondria



b



SLC17 (2.A.1)

Vesicular glutamate transporters

SLC25 (2.A.29)

Mitochondrial transporters



SLC25A 12, A13



AGC1,2



AAA



A1



Mitochondria



SLC25A 18,A22



GC2, 1



Glu



S2



Mitochondria



SLC25A29



ORNT3



Om, acylcarnitine



?



Mitochondria



SLC32A1



VGAT



GABA, Gly



A3a



Endosome



SLC36A1



PAT1 (LYAAT1)



Pro, Gly, GABA



S2



Lysosome



SLC36A4



PAT4 (LYAAT2)



Pro, Trp



S2



Lysosome



SLC38A7



SNAT7



NAA, CAA



S1/S3?



Lysosome



SLC38A9



SNAT9



Gln, Arg



S1/S3?



Lysosome



PQLC2



LAAT1



CAA



S2?



Lysosome



PQLC4



CTNS (cystinosin)



Cystine



S2



Lysosome



SLC32 (2.A.18)

Vesicular inhibitory AA

transporters

SLC36 (2.A.18)

Proton-coupled AA transporters



SLC38 (2.A.18)

Small neutral AA transporters



LCT (2.A.43)

Lysosomal cystine transporters



Cl2 dependent, exact mechanism uncertain.

H1 dependent.

First column shows transporter families as classified by HGNC and (in parentheses) IUBMB. See, for example, Hediger et al. (2013); Schioăth et al. (2013);

Jezegou et al. (2012) for more detailed information and original sources.

AAA, anionic AA; NAA, neutral AA; CAA, cationic AA.

a



b



independent of substrate translocation (these gates may be as small as a single AA residue in the transporter

protein structure), effectively locking substrate in place at the binding center during certain stages of the transport cycle (the “gated-pore” mechanism). The gated, substrate-bound states are of low energy and the reorientation of the unloaded transporter across the membrane bilayer is generally identified as the rate-limiting

energy barrier (see, eg, Forrest et al., 2011; Fotiadis et al., 2013 for detailed reviews of transport mechanisms).

AA transport may be coupled to movements of other substrates including Na1, H1, K1 and/or Cl2 (typically

Na1-AA symport, aka cotransport) as well as movement of other AA by antiport (Fig. 5.1D). Binding of multiple substrates to a transporter may follow a specific order.

Functionally, the rate of AA transport increases in proportion (determined by Km) to the cis AA concentration until the transporters approach saturation (see Fig. 5.2), at which point transport rate can only be further

increased by altering certain aspects of the driving force(s) or increasing the number of transporters (Vmax).

For AA transporters with cosubstrates (symport), cis concentration of all substrates will influence AA transport

rate. Equally, for AA antiport the trans concentration of the counter-substrate will influence cis to trans AA

transport rate.

The binding centers of most AA transporters encoded by mammalian genomes recognize a range of

structurally-similar AA as cargo for transport (see Tables 5.1 and 5.2), typically either large neutral AA (LNAA),

small neutral AA (SNAA), cationic AA (CAA), or anionic AA (AAA). Of the eight dietary essential (aka indispensable) AAs, Lys is a CAA whilst the other seven (Leu, Ile, Val, Phe, Trp, Met, Thr) are considered as LNAA

in the current context. Most AA transporters are stereoselective for the proteinogenic L-forms of AA, although

the SLC36 transporter family and SLC1A4/A5 are characterized by a relatively-low stereoselectivity and

have particular importance for transport of small, biologically-active D-AA such as D-Ser (Edwards et al., 2011).



I. GENERAL AND INTRODUCTORY ASPECTS



52



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



FIGURE 5.1 Prototypic AA transporter structure and mechanism. (A) The transporter polypeptide typically forms 10 or 12 transmembrane helixes (depending on fold type) within the membrane. IN, OUT shown in relation to the cytosol. Outward facing regions may be glycosylated. (B) The helices fold within the membrane to form a central hydrophilic translocation pathway (dotted arrow) with a localized binding

center (gray circle). For clarity, peptide regions peripheral to the membrane are not shown. (C) The Alternate Access transport mechanism.

AA transport is proposed to proceed stepwise from cis to trans compartments through a sequence of states: (1) AA adsorbs or “binds” to the

binding center of the free transporter open to the cis compartment (State A); (2) Conformational changes induced by AA binding produce

intermediate transition states, which may include gated states (gates indicated by dashed lines), such as a fully-occluded state (State B).

These result in bound AA becoming exposed at the trans membrane surface through the central translocation pathway; (3) AA is released into

the trans compartment (State C); and (4) the free transporter reorientates to the cis-facing conformation. These steps are reversible. Only three

states of uniport are shown here for simplicity. AA transport may be coupled to symport/antiport of ions and/or antiport of another AA.

Symport proceeds similarly to uniport when all substrates are bound or released. For antiport, the final reorientation step (4) only occurs after

the antiported substrate has bound to the free transporter in the trans-facing conformation. (D) Major transport-cycle stoichiometries for mammalian AA transporters, directionality shown in relation to cytosolic fluid (CF) and extracellular/endosomal fluid (EF). U, Uniport; S, cation—

AA Symport (primary Na1-coupling except for S2); A, Antiport (primary AA antiport except for A3). Transport cycles generating a net electrical charge (rheogenic mechanisms, eg, S1, S2, S5, A2 for neutral amino acids) are influenced by both chemical gradient of substrates and electrical gradient (membrane potential): for Na1-AA symport, this leads potentially to a 100-times accumulation of substrate AA per Na1

transported (Stein, 1986). Electroneutral transport cycles are only influenced by chemical gradient of substrate.



The classification of AA transporters based on genetic similarity now supersedes an earlier “Transport System”

classification based on substrate selectivity and transport mechanism (see Table 5.1), although the latter remains

useful as a functional descriptor (Broer, 2008; Hediger et al., 2013; Taylor, 2014).



5.2. AA TRANSPORTERS AND CELLULAR FUNCTION

5.2.1 Cellular Nutrient Supply

Alongside being the basic structural units for protein synthesis, many AA also function as important metabolic

intermediates and/or as substrates for biosynthesis of low-molecular weight molecules of physiological importance (see, eg, Manjarin et al., 2014; Wu, 2013 for review). Sources of cytosolic AA for these processes include

(1) the extracellular fluid, (2) proteolytic endosomes such as lysosomes, and (3) cytosolic biosynthetic/proteolytic

pathways (see Fig. 5.3). Sources (1) and (2) both require the AA to be transported into the cytosol. AA transporter

expression is tissue-specific and many cell types express several AA transporters with overlapping substrate

selectivities, such that transport of a specific AA across a cell membrane frequently involves the integrated



I. GENERAL AND INTRODUCTORY ASPECTS



5.2. AA TRANSPORTERS AND CELLULAR FUNCTION



53



FIGURE 5.2 A simple AA transport versus concentration relationship. Transport rate increases from zero to a maximum plateau value

(Vmax) as substrate concentration increases, tracing a single rectangular hyperbola. Values shown on y- and x-axes are nominal. Vmax represents the transport rate when the system has reached full capacity and Km is the substrate concentration at which transport rate is halfmaximal (0.5 Vmax). The graph shows two representative curves for different AA transporters with similar Vmax but different Km values

(20 and 165 μmol/L for black and gray curves respectively). The arrows on the x-axis indicate the ranges of AA concentration over which each

transporter is the more sensitive to changes in AA concentration.



activity of uniporters, symporters, and antiporters operating in parallel (see Fig. 5.3). The functional interactions

between AA for transport across cell membranes in vivo, which include cis-competition and (for antiporters)

trans-effects, are influenced by (and may impact on) the nutritional and physiological status of the body

(Christensen, 1990; Taylor, 2014 for review). Many cell types express both high-Km and low-Km transporters for

particular AA types (cf. Fig. 5.2). Such dual-transporter systems act to “fine-tune” sensing of nutrient depletion

through integration of inputs relating to internal and external nutrient availability and may facilitate both preparation for and recovery from cellular starvation (Levy et al., 2011).

In a typical mesodermal cell, CAA and LNAA are taken up by uniport or antiport mechanisms whereas

SNAA and AAA are transported by concentrative Na1-coupled symport (see Fig. 5.3). This results in SNAA such

as glutamine and alanine becoming highly-concentrated in cell types such as skeletal muscle, where they may

function at least partly as labile nitrogen stores (Brosnan, 2003; He et al., 2010). In contrast, LNAA do not accumulate to any great extent and indeed tend to equilibrate between intracellular and extracellular fluids.

Facilitative transport (or “facilitated diffusion”) by AA uniporters such as SLC7A1-A4 and SLC43A1-A2 provides

a route for CAA and LNAA respectively to move down a transmembrane (electro)chemical potential gradient

where simple diffusion across the lipid bilayer is energetically unfavorable. A sequential relationship between

primary, secondary, and tertiary active transport systems (P, S, T; see Fig. 5.3) contributes substantially to

transport of LNAA across cell membranes; this includes symport (cotransport) and antiport (exchange)

mechanisms operating in series downstream of the Na1 pump. This relationship hinges on the ability of a subset

of AA (notably glutamine) to be accepted as substrates by both secondary and tertiary active AA transporters.

The best-studied of these AA transporters are SLC38A2, SLC1A5 (both secondary active transporters mediating

Na1-glutamine symport), and SLC7A5 (an LNAA antiporter mediating tertiary active transport when acting

downstream of either symporter) (Baird et al., 2009; Nicklin et al., 2009; Sinclair et al., 2013). In epithelial cells,

broad-scope Na1-AA symporters, including SLC6A19 (Broer et al., 2011) and SLC6A14 (Van Winkle et al., 2006),

enable all types of AA to be transported directly into cells.

AA transport from extracellular fluid appears not to be a limiting source of AA for the cytosolic pool used

in cellular mRNA translation (protein synthesis) in quiescent or terminally differentiated cells, but may

become a more important factor in rapidly growing and proliferating cells as well as those with high rates of

protein secretion. Notably, upregulation of plasma membrane AA transport is a characteristic feature of cell

activation and transformation (often driven by growth factor signaling; McCracken and Edinger, 2013), thereby

increasing intracellular availability of AA for protein synthesis and/or cell metabolism. Furthermore, rapid

growth/proliferation of mammalian cells is inhibited by genetic or functional inactivation of specific AA transporters (eg, Heublein et al., 2010; Nicklin et al., 2009; Sinclair et al., 2013; Usui et al., 2006). Lysosomal AA

transport mechanisms are poorly studied but represent a key step in the recycling of AA between processes of



I. GENERAL AND INTRODUCTORY ASPECTS



54



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



FIGURE 5.3 AA transporters, cell metabolism, and nutrient signaling. CF, cytosolic fluid; EF, extracellular/endosomal fluid. AA may

enter (or exit) cells down chemical gradients generated by metabolic processes (eg, protein synthesis (PS)/breakdown (PB), AA biosynthesis

(A), AA catabolism (C)) either through facilitative transport (F, a uniport mechanism) or exchange (E, an antiport mechanism). Charged AA

movements will also depend on electrical gradient. AA may be accumulated in the CF from the extracellular fluid against an (electro)chemical

gradient, downstream of primary active transport (P, the Na1-K1 ATPase pump at the plasma membrane), by secondary active transport

(S, Na1-AA symport mechanism using the Na1 electrochemical gradient generated by P) or by tertiary active transport (T, AA antiport

mechanism using AA1 gradient generated by S to drive accumulation of AA2). AA transporters on endosomal membranes (eg, the H1-AA

transporter SLC36A1 and the lysosomal cystine transporters (LCT)) more typically utilize an H1 gradient generated by the v-ATPase

H1 pump to drive AA movement from endosomal fluid into the CF. AA delivered to the CF by AA transporters may directly modulate

transcription factor activity in the cell nucleus, for example, the SLC7A5 substrates T3 and T4 are ligands for Thyroid Receptors (TR) and

kynurenine is a ligand for Aryl Hydrocarbon Receptors (AHR). AA transporter activity contributes to maintenance of free AA concentrations

in CF: fluctuations in cytosolic AA concentrations indirectly modulate abundance of transcription factors such as ATF4 (which binds to AARE

on AA-responsive genes). AA may also move from CF into the lysosomal lumen through transporters such as SLC7A5 and SLC38A9

(the exact nature of substrate coupling for SLC38A9 is not clear at present). Fluctuations in intralysosomal AA concentrations appear to be

sensed by SLC38A9 as part of the mechanism for recruitment and subsequent activation (denoted *) of the mTORC1 signaling complex at the

lysosomal membrane. The RagÀRagulator (Rgltr) protein complexes act as scaffold/signaling elements. Rheb activates mTORC1 on the

lysosome and is a focal point for integration of upstream AA and growth factor signals. AA metabolites such as amines (eg, dopamine,

tryptamine) and NO (a product of Arg metabolism) may also act as intra- or extracellular signaling molecules.



protein synthesis and breakdown and may be of particular importance for maintaining protein mass of cells at

steady-state (eg, Liu et al., 2012).

AA transport at the cell surface may also be increased for scavenging purposes during periods of AA deprivation. This process, known as “adaptive regulation,” is exemplified by SLC38A2 and involves transcriptional upregulation of transporter gene expression via translational upregulation of activating transcription factor 4 (ATF4)

(a transcription factor which binds AA response elements (AARE) in target genes such as SLC38A2, activating

transcription) (Palii et al., 2006), maintenance of SLC38A2 mRNA translation through an internal ribosome entry

site (Gaccioli et al., 2006) and increased stability (reduced degradation) of SLC38A2 transporter proteins (eg,

Hyde et al., 2007).



5.2.2 Nutrient Sensing

Nutrient-responsive signaling pathways involved in control of cell growth, proliferation, and metabolic rate

utilize a variety of enzymes, receptor and transporter proteins as nutrient sensors (Duan et al., 2015; Kimball,

2014; Taylor, 2014 for review). The major AA sensing-signaling pathways in mammalian cells are the GCN

(general control nonderepressible) and mTORC1 (mammalian/mechanistic target of rapamycin complex 1)

pathways. The GCN pathway is activated when one or more AA are scarce and primarily senses cytosolic AA



I. GENERAL AND INTRODUCTORY ASPECTS



5.2. AA TRANSPORTERS AND CELLULAR FUNCTION



55



availability at the level of AA “charging” on tRNA (Gallinetti et al., 2013; Sonenberg and Hinnebusch, 2009); its

activation inhibits global protein synthesis. The mTORC1 pathway is activated when certain AA (eg, leucine, glutamine, arginine) are abundant and includes sensors which monitor AA availability in both cytosol and subcellular organelles such as lysosomes (Duan et al., 2015; Kim et al., 2013); mTORC1 activation promotes net protein

accretion by simultaneously stimulating protein synthesis and inhibiting autophagic protein breakdown

(Kimball, 2014 for review). Several putative cytosolic AA sensors linked to mTORC1 activation have been

reported, including some capable of directly binding AA such as leucine (eg, leucyl-tRNA synthetase) (Han et al.,

2012; Kim et al., 2013), alongside membrane-bound AA sensors (Duan et al., 2015 for review). AA transporters

are now recognized to have important roles in both sensor and effector arms of the GCN and mTORC1 pathways

(see Kim et al., 2013; Kriel et al., 2011; Taylor, 2014 for review). AA transporters may act directly as the initiating

sensor for a signaling pathway (see Section 5.2.2.1) or serve as a conduit for delivery of extracellular AA to intracellular AA sensors (see Section 5.2.2.2). They may also generate indirect nutrient-related signals related to effects

of transported substrates on intracellular pH and volume (Hundal and Taylor, 2009 for review).

5.2.2.1 AA Transporters as AA Sensors

AA transporter activity reflects substrate quantity (or “availability”) as functions of both binding-site occupancy and transport-cycle rate (over a particular range of substrate concentration; see Fig. 5.2) and the intrinsic

link between substrate binding and protein conformational change (see Fig. 5.1) affords them considerable potential as nutrient “sensors” (although outwith their range of sensitivity they can only qualitatively sense presence

or absence of substrate for a signaling mechanism unless the gain of the transduction mechanism is modulated).

Furthermore, AA transporters are uniquely positioned to access and “sample” both cytosolic and extracytosolic

(extracellular/endosomal) AA pools. Mammalian AA transporters such as SLC38A2/A9 and SLC36A1/A4 may

also act as AA-sensing receptors (ie, AA substrate binding to the transporter protein induces an intracellular

nutrient signal independent of AA transport) and thus act as multifunctional AA transceptors on either endoă gmundsdottir et al., 2012; Pinilla et al., 2011; Rebsamen et al., 2015; Wang et al.,

somal or plasma membranes (O

2015). The AA-dependent recruitment of the mTORC1 signaling complex to lysosomal membranes (a key stage

in mTORC1 activation) is associated with AA accumulation into lysosomes (Zoncu et al., 2011). A lysosomalanchored “nutrisome” protein complex appears to act as a sensor of intralysosomal AA levels upstream of

ă gmundsdottir et al.,

mTORC1 activation. Both SLC38A9 and SLC36A1 are included as part of the “nutrisome” (O

2012; Rebsamen et al., 2015; Wang et al., 2015) alongside the v-ATPase which helps develop the acidic lumen of

the lysosome (pH 5) by actively pumping H1 from the cytosol (see Fig. 5.3). Detection of influx, efflux, and/or

accumulation of AA into the lysosomal lumen by the nutrisome (a so-called “inside-out” method of AA sensing;

Rebsamen et al., 2015; Zoncu et al., 2011), causes activation of the guanine exchange factor function of the

Ragulator complex which in turn promotes the guanosine triphosphate charging of RagA/B subunits necessary

for mTORC1 engagement and activation (see Fig. 5.3; see Duan et al., 2015 for review). SLC38A9 accepts both

glutamine and arginine as substrates (Rebsamen et al., 2015; Wang et al., 2015) and an AA-dependent conformational change of SLC38A9 involving its cytosolic N-terminus (a domain binding directly to Ragulator) may

directly initiate an AA signal through this transceptor (Wang et al., 2015). Transceptor signaling is likely to be initiated during substrate-occluded states of the transport cycle (see Fig. 5.1; see Kriel et al., 2011; Taylor, 2014 for

review). SLC38A7 is another SLC38-family transporter localized to lysosomal membranes (Chapel et al., 2013)

and it appears to accept a broad range of neutral and CAA as substrates (Haăgglund et al., 2011). The SLC38family AA transporters are highly pH-sensitive and a subset operate as Na1-dependent H1-AA antiporters (see

Schioăth et al., 2013 for recent review), a mechanism which might favor glutamine and arginine accumulation into

lysosomes by both SLC38A7 and A9. The requirement for lysosomal localization of mTORC1 for its activation

may differ between leucine and other AA (Averous et al., 2014). Nevertheless, lysosomal AA transport mechanisms resembling the L and T Systems for LNAA in plasma membranes have been characterized functionally

(Andersson et al., 1990; Pisoni and Thoene, 1991; Stewart et al., 1989) which could mediate net lysosomal uptake

or release of essential LNAA depending upon cellular AA status. Indeed very recent findings (Milkereit et al.,

2015) indicate that SLC7A5 (as the functional SLC7A5—SLC3A2 System L heterodimer) appears to be recruited

constitutively to lysosomes by the lysosomal-associated transmembrane protein 4b, where it promotes mTORC1

activation via the v-ATPase nutrisome complex. As an AA antiporter, SLC7A5 would require a sufficiency of

exchangeable AA in the lysosomal lumen (produced by proteolysis and/or uptake through other AA transporters) in order for it to accumulate leucine into this compartment. SLC36A1 may exert a negative influence on

lysosomal mTORC1 signaling by mediating H1-dependent efflux of SNAA from the lysosomal lumen into the

cytosol (Zoncu et al., 2011). PQLC2 is a lysosomal exporter of CAAs (Je´ze´gou et al., 2012; Liu et al., 2012).



I. GENERAL AND INTRODUCTORY ASPECTS



56



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



The “inside-out” lysosomal AA sensing mechanism implies sensing of AA availability in the lysosomal lumen,

which for a membrane-bound transceptor such as SLC38A9 is spatially equivalent to the extracellular space.

Another SLC38 family member, SLC38A2, functions as a transceptor at the plasma membrane linking extracellular AA availability to cell function by a mechanism also involving its cytosolic N-terminal protein domain

(Hyde et al., 2007; Pinilla et al., 2011). Characteristic properties of cell-surface nutrient transceptors in lower

eukaryotic organisms include (1) substrate transport/binding promotes internalization and degradation of the

transceptor protein when substrate is abundant and (2) induction of transceptor gene expression in cells starved

of their substrates (Conrad et al., 2014; Kriel et al., 2011). SLC38A2 displays both these transceptor properties

(Hyde et al., 2007) and its upregulation as an AA scavenger during periods of AA deficiency is an important

effector arm of the GCN pathway in mammalian cells.

5.2.2.2 AA Transporters Upstream of Intracellular AA Sensors

AA transporters at the plasma membrane deliver extracellular AAs to the intracellular sensor molecules associated with mTORC1 and GCN pathway regulation. Changes in cell-surface AA transporter expression or extracellular AA concentration (the latter within relative limits highlighted in Fig. 5.2) will alter these rates of AA

delivery, which may influence nutrient-dependent signaling. SLC7A5 appears to be of particular importance for

leucine-dependent activation of mTORC1 and may provide a direct, two-stage route for leucine movement

between extracellular and lysosomal compartments (Milkereit et al., 2015; see Fig. 5.3). The level of functional

cell-surface SLC7A5 expression in fibroblasts correlates directly with the effectiveness of leucine-induced

mTORC1 activation in these cells (Schriever et al., 2012). Induction of functional SLC7A5 gene expression is an

initiating factor for mTORC1 pathway activation by the proliferation factor HIF2α in vivo (Elorza et al., 2012)

and for mTORC1-dependent T-lymphocyte activation (see Section 5.5); induction of the functionally similar

SLC7A8 has also been linked to cell proliferation in vivo (Kurayama et al., 2011). Leucine transport by SLC7A5/

A8 requires obligatory AA antiport, typically leucine-glutamine antiport. The ability of glutamine transporters

such as SLC38A2 and SLC1A5 to develop or maintain a suitable large intracellular glutamine pool for antiport

may become limiting for leucine-dependent mTORC1 activation downstream of SLC7A5 (Baird et al., 2009;

Nakaya et al., 2014; Nicklin et al., 2009). SLC7A5/SLC3A2 and SLC1A5 have been reported (Xu and Hemler,

2005) to colocalize with CD147 as part of a cell-surface protein “supercomplex” linked to activation of cell

metabolism and proliferation.

Delivery of extracellular arginine via the SLC7A1 and A3 transporters may also influence mTORC1 pathway

activation (Huang et al., 2007).



5.2.3 Cell-Cell Communication

The SLC1 high-affinity glutamate transporters have key functions in the control of excitatory neurotransmission

within the central nervous system (CNS) (Kanai et al., 2013), principally in the clearance of neurotransmitter AA

from the synaptic cleft, whilst the SLC6 family transporters for gamma-aminobutyric acid (GABA) and glycine have

similar roles in inhibitory neurotransmission (Pramod et al., 2013). The neurotransmitter AA are concentrated

within neuronal synaptic vesicles by vesicular AA transporters (SLC17/32 transporter families; Table 5.2) which utilize the endosomal pH gradient (see Fig. 5.3) as a driving force. The SNAA transporter SLC7A10 (which is unusual

in that it can operate by either antiport or uniport mechanisms) acts in the CNS to regulation synaptic concentration

of NMDA (glutamate) receptor coagonists D-serine and glycine (Xie et al., 2005).

Certain AA transporters have specific roles in transport of biologically-relevant AA derivatives involved in

intercellular signaling, for example, SLC7A5 accepts thyroid hormones and L-DOPA as substrates (Taylor and

Ritchie, 2007). SLC7A2 and SLC7A7 have opposing roles in the regulation of intracellular levels of the NO

precursor arginine (Ogier de Baulny et al., 2012; Yeramian et al., 2006).



5.3. AA TRANSPORTERS IN WHOLE-BODY NUTRITION

5.3.1 Absorption of AA and Peptides

The recommended daily allowance for dietary protein intake by an adult person is 0.8 g/kg per day, irrespective of overall energy intake (Layman et al., 2015). Dietary protein of high quality includes sufficient amounts of

all essential AA required for a particular stage of life (Wu, 2013). This exogenous protein (along with a similar



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57



5.3. AA TRANSPORTERS IN WHOLE-BODY NUTRITION



TABLE 5.3



Peptide transporters



Gene family



HUGO



Acronym



Substrates



Mechanism (see Fig. 5.1)



SLC15A1, A2



PEPT1, 2



Tri- and di-peptides, β-lactams



S2



SLC15A3, A4



PHT2, 1



Tri- and di-peptides, Histidine



S2



SLC15 (2.A.17)

Peptide transporters



First column shows transporter families as classified by HGNC and (in parentheses) IUBMB. See, for example, Hediger et al. (2013); Gilbert et al. (2008) for more

detailed information and original sources.



FIGURE 5.4 Intestinal digestion and absorption of amino acids and peptides. Dietary proteins are digested by hydrolysis (proteolytic

enzymes indicated E in Figure) to absorbable peptides (shown as AA-AA) and amino acids (AA). Peptides (di/tripeptides) are absorbed into

intestinal epithelial cells across the brush-border membrane (BBM) by H1 coupled transport using the H1 gradient produced by the acid

microclimate exterior to the BBM. Most of these peptides are hydrolyzed to AA within the epithelial cells. The major BBM transport system

for neutral AA is the Na1 coupled transporter SLC6A19 (B0AT1). The major transporters for cationic and anionic AA at the BBM are SLC7A9

(b0,1AT) and SLC1A1 (EAAT3) respectively. Additional BBM transporters for neutral AA (eg, SLC1A5, SLC36A1, SLC6A20) are not shown on

Figure. AA antiporters (notably SLC7A6 and SLC7A8) predominate at the basolateral membrane (BLM), enabling physiologically-useful AA

exchanges which, in general, are osmotically-neutral. Facilitative transport of neutral AA at the BLM appears to be mediated by SLC43A2 and

(not shown) SLC16A10. Note (1) targeting of SLC7 antiporters to either BBM or BLM by SLC3A1 or SLC3A2 subunits respectively, (2) cycling

of NAA at both membranes as part of the mechanism to absorb CAA by AA antiport, and (3) cellular uptake of cystine (Cys; metabolized to

cysteine inside cells) by AA antiporters at both membranes.



quantity of endogenous protein released into the digestive tract) is hydrolyzed in the gut lumen by digestive proteases to absorbable constituents which include di- and tripeptides as well as AAs (see, eg, Goodman, 2010 for

review). The products of protein digestion must then be moved across both the brush-border membrane and

basolateral membrane of the intestinal epithelium (BBM, BLM respectively; see Fig. 5.4) in order to be absorbed

by the body. Digestive enzymes include endopeptidases (eg, pepsin, trypsin) and exopeptidases (eg, carboxypeptidase A) secreted by the gastrointestinal tract, as well as numerous BBM-bound and cytosolic enzymes (including aminopeptidases). Active AA and peptide absorption is driven by Na1 and H1 electrochemical gradients

established by the Na1/K1 ATPase pump at the epithelial BLM (see Fig. 5.4) and Na1/H1 antiporters at the

BBM. Peptides are absorbed by H1-coupled symport (Table 5.3, Fig. 5.4) and are largely hydrolyzed to AA in the

epithelial cells, rather than being absorbed intact into the bloodstream (Gilbert et al., 2008; Nakashima et al.,

2011). Neutral and AAA are taken up into intestinal epithelial cells by Na1-coupled symport (principally by

SLC6A19 and SLC1A1 respectively), whereas CAA are transported largely by AA antiport (SLC7A9). All AA

pass from the epithelial cells to the bloodstream by AA antiport and/or facilitative transport (uniport) systems at



I. GENERAL AND INTRODUCTORY ASPECTS



58



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



the BLM. Notably, glutamine/essential NAA antiport at the BLM via SLC7A8 has the dual benefit of providing

glutamine as an intestinal fuel and completing essential NAA absorption. SLC7A6 mediates electroneutral AA

exchanges (balancing Na1 and CAA positive charges) which tend to favor efflux of CAA from cells. The BLM

transport step is typically rate-limiting for AA absorption.

The relative importance of peptide and free AA absorption at the BBM depends upon the digestibility of the

ingested protein (Gilbert et al., 2008; Nakashima et al., 2011). Peptide transport facilitates absorption of highprotein loads in the intestine (Nassl et al., 2011), although it does not appear to be nutritionally-essential

(Hu et al., 2008). The SLC6A19 AA transporter forms functional “digestive complexes” with specific peptides (eg,

aminopeptidase N) in the BBM which improve its absorptive efficiency (Fairweather et al., 2012). SLC6A19 provides an important AA supply for epithelial mTORC1 signaling and SLC6A19 knockout mice exhibit a phenotype

of apparent epithelial cell starvation (Broer et al., 2011). Inherited disorders of epithelial AA absorption include

AA transport defects at both BBM; for example, cystinuria (SLC7A9/SLC3A1 defect), Hartnup disorder (SLC6A19

defect) and BLM; lysinuric protein intolerance (SLC7A6 defect) (see Broer and Palacin, 2011 for review). These

disorders of AA absorption rarely result in any specific AA deficiency because of the overlapping substrate selectivities of intestinal AA transporters.

The absorptive capacity for protein (as AA and peptides) is regulated in relation to both quantity and quality

of dietary protein and is generally maintained at a higher level than the normal dietary supply (thus providing a

“safety factor” for efficient absorption of high-protein meals) (Diamond and Hammond, 1992). Intestinal AA

transporter expression may be induced by raised levels of dietary protein or free AA mixtures, but also by protein intakes below those required for essential AA balance to ensure supply of these nutrients (Buddington et al.,

1991). Several endocrine factors may be involved in this type of regulation. Epidermal growth factor (EGF)

facilitates intestinal growth and adaptation and signaling downstream of EGF may activate functional expression

of SLC6A19 (Bhavsar et al., 2011). The carboxypeptidase ACE2 (a peptidase interacting with SLC6A19 at the

intestinal BBM; Fairweather et al., 2012) may also be involved in regulation of its functional capacity for AA

transport (Vuille-Dit-Bille et al., 2015). Leptin upregulates expression and transport capacity of the peptide transporter SLC15A1 in the intestine when secreted into the gut lumen (Buyse et al., 2001), although abundance of AA

transporters (SLC6A19, SLC1A5) in the BBM of intestinal cells is downregulated by leptin (Ducroc et al., 2010;

Fanjul et al., 2012). To add further complexity, rapid regulation of intestinal AA transport activity in vivo may

involve local neural circuits (Mourad et al., 2009). SLC38A2 and the SLC15A1 peptide transporter may form part

of a nutrient-sensing system in the gastrointestinal tract which causes gut hormone release upon activation

(Young et al., 2010; Zietek and Daniel, 2015), indeed an oral AA load has been shown to exert an incretin effect

mediated by glucose-dependent insulinotropic polypeptide (Lindgren et al., 2015). External to the gut itself,

SLC6A15 forms part of an AA sensor in the hypothalamus involved in regulation of food intake (Drgonova et al.,

2013; Hagglund et al., 2013). Similarly, SLC38A2 is a secondary component of an AA-sensing system in the

anterior piriform cortex of the brain which allows animals to make rapid food selections on the basis of essential

AA quality (Gietzen and Aja, 2012).



5.3.2 Interorgan Nitrogen Flow

AA transporter capacity and competition between different AA for transport at the bloodÀtissue interface are

important factors in the determination and regulation of AA flows between mammalian tissues (see, eg, Brosnan,

2003; Christensen, 1990; van de Poll et al., 2004 for review).

In the fed state, the predominant AA flows are from the intestine to other tissues and AA in excess of those

required for protein synthesis or particular metabolic purposes are rapidly catabolized, typically as oxidative

fuels. Excess nitrogen is excreted as urea: the urea-cycle intermediates arginine and ornithine are provided to

hepatocytes (which unusually do not express SLC7A1) at least partly by SLC7A2 (Closs et al., 2006). Blood AA

concentrations increase during the absorptive phase, indicating that the rate of intestinal AA absorption

exceeds the capacity for tissue AA uptake. The increase in essential AA availability after protein or AA ingestion upregulates expression of AA transporters (eg, SLC38A2, SLC7A5) in skeletal muscle 2À3 h postmeal

(Drummond et al., 2010), an adaptive response to help increase intracellular delivery of AA (and thereby

enhance the mTORC1 growth signal from nutrients and insulin) during this anabolic phase of the dietary cycle

(see Kimball, 2014; Layman et al., 2015 for review). The strong pH-dependence of SLC38A2 makes it very

sensitive to inhibition by plasma acidification and this feature contributes to the protein-catabolic effects of

metabolic acidosis (Evans et al., 2008).



I. GENERAL AND INTRODUCTORY ASPECTS



5.5. AA TRANSPORTERS AND THE IMMUNE RESPONSE



59



In the postabsorptive and fasted states, the dominant AA flows are between muscle, liver, and kidney and

these assist in conserving body nitrogen stores by channeling protein and AA catabolism to production of glutamine and alanine rather than urea (Brosnan, 2003; van de Poll et al., 2004). Within the liver, perivenous hepatocytes take up glutamate through SLC1A2 for synthesis of glutamine, which is released into the bloodstream by

the Na1-H1-antiporting SLC38A3 transporter operating unusually in reverse mode (under circumstances where

the combined glutamine and H1 gradients apparently exceed the inwardly-directed Na1 gradient) (Baird et al.,

2004; Broer et al., 2002). Expression of SLC1A1 in kidney, muscle, and lung provides glutamate for glutamine

synthesis in these tissues. This glutamine can be exchanged for essential NAA through plasma membrane AA

antiporters (eg, SLC7A5 and A8) in tissues such as skeletal muscle, providing essential NAA to sustain muscle

protein synthesis whilst simultaneously increasing glutamine availability in the blood for other tissues to use

either as a fuel (eg, lymphocytes, intestinal epithelium) or as an aid to maintaining optimum acidÀbase and nitrogen stasis (eg, kidney) (Baird et al., 2004; He et al., 2010; Karinch et al., 2007). SLC38A3 is upregulated in the kidney during circumstances of metabolic acidosis to increase glutamine uptake from the blood as a source of NH41

for urinary acid excretion (Moret et al., 2007). The essential NAA uniporter SLC43A1 is upregulated by starvation

in both liver and skeletal muscle (Fukuhara et al., 2007). This would tend to favor AA efflux from these tissues

into the bloodstream and might participate in the supply of essential NAA to organs such as the brain during

prolonged starvation. Alanine is an important carbon source for hepatic gluconeogenesis in fasted states and

SLC38A2 is upregulated (under the influence of glucagon) in periportal hepatocytes to help maintain alanine

supply from the bloodstream under such circumstances (Varoqui and Erickson, 2002).



5.4. AA TRANSPORTERS IN MAMMALIAN EMBRYONIC

DEVELOPMENT AND GROWTH

Several AA transporters are essential for mammalian embryonic development (eg, SLC7A5; Poncet et al., 2014)

or perinatal survival (eg, SLC7A1; Perkins et al., 1997; SLC43A2; Guetg et al., 2015). This may reflect either a particular importance for AA supply over these periods or a specific function in processes such as signaling of

growth or early tissue differentiation.

AA activation of mTORC1 is an important aspect of blastocyst activation during early stages of development

(Gonzalez et al., 2012). Upregulation of SLC6A14 (a broad-scope Na1/Cl2-coupled AA transporter) at the blastocyst stage enhances uptake of AA which helps promote blastocyst activation and trophoblast outgrowth (Van

Winkle et al., 2006). Placental AA transporters supply the growing fetus with AA from the maternal bloodstream

(see, eg, Dilworth and Sibley, 2013 for review). Placental growth is also modulated by mTORC1 which regulates

the expression and activity of key AA transporters (eg, SLC7A5) at the placental surface (Roos et al., 2009).

Intrauterine growth restriction is associated with decreased activity of placental AA transporters in conjunction

with reduced placental mTORC1 activity (Roos et al., 2007). During normal pregnancy, expression of AA transporters including the essential NAA uniporters SLC16A10 and SLC43A1 correlates positively with birth-size of

infants (Cleal et al., 2011). In contrast, an inverse correlation between SLC38A2 expression and placental weight

is observed; that is, smaller placentas appear to upregulate SNAA uptake by this particular route (Coan et al.,

2008). This may help to maintain fetal growth rate at least partly independent of placental size.

The high growth rate of newborn and infant tissues (particularly skeletal muscle) is maintained in part by AAdependent stimulation of the mTORC1 pathway which promotes net protein synthesis (see Suryawan and Davis,

2011 for review). Maternal milk provision for growing infants requires extremely high rates of protein turnover

in lactating mammary glands. AA such as lysine, methionine, and tryptophan may become limiting for milk production, at least in livestock diets (see Manjarin et al., 2014 for review). AA transporters including SLC7A1,

SLC7A5, SLC6A14, and SLC38A2 are upregulated correspondingly in lactating mammary tissue, correlated with

the action of hormones such as prolactin and oestradiol (Aleman et al., 2009; Manjarin et al., 2011; VelazquezVillegas et al., 2015).



5.5. AA TRANSPORTERS AND THE IMMUNE RESPONSE

AA transporters have functional importance for cells of both the innate and adaptive immune systems.

SLC7A2 is induced by activation in macrophages, providing arginine as a substrate for NO or polyamine



I. GENERAL AND INTRODUCTORY ASPECTS



60



5. ROLE OF AMINO ACID TRANSPORTERS IN PROTEIN METABOLISM



production depending upon type of activation (Yeramian et al., 2006). SLC7A11 (which mediates electroneutral

glutamate/anionic cystine exchange) is also upregulated in macrophages to support synthesis of the antioxidant

glutathione (Conrad and Sato, 2012). T-lymphocytes undergo periods of rapid cell growth, proliferation and protein (eg, cytokine) secretion during an immune response, following activation through the T-cell receptor. During

these periods of markedly increased protein synthesis, intracellular availability of both LNAA (eg, leucine for

protein synthesis) and SNAA (eg, glutamine or glycine for cell metabolism) may become increasingly dependent

on the AA transport capacity at the cell surface. T-lymphocyte activation is associated with a high fold-induction

of SLC7A5 which increases high-affinity (low μM Km) LNAA supply (Sinclair et al., 2013). Removal of a block to

mRNA elongation may stimulate induction of SLC7A5 (and SLC3A2) gene transcripts during T-cell activation

(Nii et al., 2001). Other AA transporters (notably SLC7A1 and SLC1A5) are also upregulated to a lesser extent

during activation of T-cells (Nakaya et al., 2014; Sinclair et al., 2013). Sustained uptake of leucine (and other

LNAA) through SLC7A5 in activated T-lymphocytes is required for mTORC1 activation, induction of the c-Myc

mitogen, and upregulation of energy-supplying metabolic pathways (Sinclair et al., 2013). The Trp metabolite

kynurenine (Kyn) is an SLC7A5 substrate and Trp/Kyn exchanges have been implicated in immunoregulatory

mechanisms and immune-escape strategies, partly linked to activation of the Aryl Hydrocarbon Receptor (AHR)

by Kyn (Kaper et al., 2007; Ramsay and Cantrell, 2015).



5.6. AA AND PEPTIDE TRANSPORTERS AS THERAPEUTIC TARGETS

Several AA and peptide transporters are of pharmaceutical interest as drug targets (for specific diseases or as

cell-growth/proliferation inhibitors) and/or as drug-delivery systems (see, eg, Hediger et al., 2013 for recent

review). Glutamate, GABA and glycine transporters of the SLC1 and SLC6 transporter families are important

pharmacological targets for neuromodulatory therapies (see Kanai et al., 2013; Pramod et al., 2013 for review).

The SLC7A5 transporter is recognized as a potential immunosuppressive (Usui et al., 2006) and antitumor (Oda

et al., 2010) target and high-affinity (nM Km) specific inhibitors of SLC7A5 are under development (eg, Oda et al.,

2010; Usui et al., 2006). AA transporter-specific positron emission tomography tracers are now in use for tissue

and tumor imaging in vivo (eg, Wiriyasermkul et al., 2012).

Essential NAA (especially Leu) are required for full activation of mTORC1 signaling downstream of growth

factors, such as insulin, and both SLC6A19-null mice (Jiang et al., 2015) and muscle-specific SLC7A5-null mice

(Poncet et al., 2014) show evidence of reduced baseline insulin-sensitivity, highlighting important links between

AA transporter function and endocrine control of metabolism. Leucine may also stimulate increases in tissue oxidative capacity (Sun and Zemel, 2009; Vaughan et al., 2013). Dietary leucine is therefore under evaluation as an

adjunct treatment for insulin resistance related to obesity (eg, Adeva et al., 2012; Macotela et al., 2011), although

there is controversy over the possible long-term metabolic consequences of resultant AA imbalances in vivo

(Layman et al., 2015 for review).



Acknowledgment

Sources of Funding: PMT acknowledges research funding from The Wellcome Trust, Biotechnology and Biological Sciences Research Council

UK, Diabetes UK, National Institutes of Health USA, and the Rural and Environment Science and Analytical Services Division (RERAD) of

The Scottish Government.



References

Adeva, M.M., Calvino, J., Souto, G., Donapetry, C., 2012. Insulin resistance and the metabolism of branched-chain amino acids in humans.

Amino Acids 43, 171À181.

Aleman, G., Lopez, A., Ordaz, G., Torres, N., Tovar, A.R., 2009. Changes in messenger RNA abundance of amino acid transporters in rat mammary gland during pregnancy, lactation, and weaning. Metabolism 58, 594À601.

Andersson, H.C., Kohn, L.D., Bernardini, I., Blom, H.J., Tietze, F., Gahl, W.A., 1990. Characterization of lysosomal monoiodotyrosine transport

in rat thyroid cells. Evidence for transport by system h. J. Biol. Chem. 265, 10950À10954.

Averous, J., Lambert-Langlais, S., Carraro, V., Gourbeyre, O., Parry, L., B’Chir, W., et al., 2014. Requirement for lysosomal localization of

mTOR for its activation differs between leucine and other amino acids. Cell. Signal. 26, 1918À1927.

Baird, F.E., Beattie, K.J., Hyde, A.R., Ganapathy, V., Rennie, M.J., Taylor, P.M., 2004. Bidirectional substrate fluxes through the system N

(SNAT5) glutamine transporter may determine net glutamine flux in rat liver. J. Physiol. 559, 367À381.

Baird, F.E., Bett, K.J., MacLean, C., Tee, A.R., Hundal, H.S., Taylor, P.M., 2009. Tertiary active transport of amino acids reconstituted by coexpression of system A and L transporters in Xenopus oocytes. Am. J. Physiol. Endocrinol. Metab. 297, E822À829.



I. GENERAL AND INTRODUCTORY ASPECTS



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