The conserved sequence NXX[S/T]HX[S/T]QDXXXT of the lactate/pyruvate:H⁺ symporter subfamily defines the function of the substrate translocation pathway

Figures & data

Table I.  Sequence of the forward primers used in the JEN1 gene mutagenesis.

Mutation	Wt codon	Mutant codon	Primers
N379Q	aat	caa	5′-gttttattggtgggtccacaatacttgactcatgcttc-3′
H383K	cat	aaa	5′-ccaaattacttgactaaagcttctcaagacttg-3′
Q386N	caa	aat	5′-cttgactcatgcttctaatgacttgttgccaacc-3′
D387E	gac	gaa	5′-ctcatgcttctcaagaattgttgccaaccatg-3′
T391S	acc	tcc	5′-caagacttgttgccatccatgctgcgtgccc-3′
N379A	aat	gct	5′-gttttattggtgggtccagcttacttgactcatgcttc-3′
H383A	cat	gct	5′-ccaaattacttgactgctgcttctcaagacttg-3′
Q386A	caa	gca	5′-cttgactcatgcttctgcagacttgttgccaacc-3′
D387A	gac	gcc	5′-ctcatgcttctcaagccttgttgccaaccatg-3′
T391A	acc	gcc	5′-caagacttgttgccatgctgcgtgccc-3′
H383D	cat	gat	5′- ccaaattacttgactgatgcttctcaagacttg-3′
D387H	gac	cat	5′-ctcatgcttctcaacatttgttgccaaccatg-3′

Figure 1.  (A) Identification of the conserved sequence ³⁷⁹NXX[S/T]HX[S/T]QDXXXT³⁹¹ (numbering refers to Jen1p) in the lactate/pyruvate:H⁺ symporter subfamily (TC#2.A.1.12.2). Multiple sequence alignment of Jen1p homologues available in the databases and the E. coli sialate transporter (NanT) was build by the Multalin (INRA) bioinformatics tool. (http://prodes.toulouse.inra.fr/multalin/multalin.html) (Corpet, 1988). (B) Predicted topology of Jen1p, built by the TMHMM software (http://www.cbs.dtu.dk/services/TMHMM-2.0/), with the location of the identified motif highlighted. The protein topology shown is composed of 12 transmembrane helices and cytoplasmic N- and C-tails.

Figure 2.  Evaluation of the uptake rate of labelled lactate (▴), pyruvate (▪) and acetate (▾) (60 µM final concentration), in the strain S. cerevisiae W303-1A pDS-1. Cells were cultivated in glucose until mid-exponential growth phase, washed and transferred to YNB lactic acid for 4 h.

Figure 3.  Epifluorescence microscopy localization of Jen1-GFP fluorescence in (A) strain jen1Δ ady2Δ transformed with the plasmids pDS-1-GFP or pUG35 and (B) isogenic Jen1 mutant strains D3987H, D387A, H383K, H383D, N379A and N379Q. S. cerevisiae strains at mid-exponential growth phase were shifted under derepressed conditions for Jen1p expression (YNB lactate 0.5% (v/v), pH 5.0) for 4 h and observed under an epifluorescence microscope and in bright field. Additionally, for S. cerevisiaejen1Δ ady2Δ pUG35 strain methionine was added for repression conditions.

Figure 4.  Relative transport rate of labelled lactate by the Jen1p mutants. The graphic represents the percentage of the initial uptake rate of the mutants considering that the initial uptake rate of the wild-type gene (pDS-1) was 100%.

Table II.  Relative transport capacity (%) of lactate, pyruvate and acetate of the Jen1p mutants, compared to the wild-type (pDS-1).

	Relative transport capacity (%)
	Lactate	Pyruvate	Acetate
p416GPD	<1	<1	0
pDS-1	100±9.0	100±10.1	100±4.2
N379Q	4±0.1	1±0.2	14±1.7
N379A	< 1	2±0	< 1
Q386N	61±0.4	46±7.5	107±3.1
Q386A	140±25.2	117±20.3	26±1.1
T391S	76±2.6	60±10	60±0.1
T391A	72±4.2	44±7.3	47±1.0
D387E	19±3.7	5±2.1	36±4.4
D387A	<1	6±1.8	18±0.7
D387H	2±1.0	<1	7±0.1
H383K	<1	<1	2±0.0
H383A	1±0.5	3±1.1	41±3.6
H383D	1±0.1	<1	18±1.1
H383D D387H	<1	1±0.5	90±5.0

Table III.  Km values (mM) of Jen1p mutants for the uptake of lactate, acetate, and pyruvate.

Allele	Km Lactate (mM)	Km Pyruvate (mM)	Km Acetate (mM)
pDS-1	0.2±0.1	0.7±0.2	4.8±1.2
Q386N	2.7±0.3	3.0±0.4	13.7±1.3
Q386A	0.2±0.1	0.3±0.1	n.d.
T391S	0.5±0.1	4.4±0.5	12.0±0.9
T391A	0.5 ±0.1	n.d.	n.d.
D387E	n.d.	n.m.	11.5±1.1
H383D D387H	n.m.	n.m.	4.5±0.7

n.d., not determined; n.m., not measurable.

Table IV.  Ki values (mM) of Jen1p mutants for propionate, benzoate, salicylate and butyrate.

	Ki values (mM)
Inhibitor	wild-type	Q386N	Q386A	T391S	T391A
Propionate	1.6±0.1	2.8±0.2	3.4±0.3	2.7±0.4	2.6±0.4
Benzoate	0.7±0.1	2.2±0.1	2.3±0.3	2.5±0.2	1.9±0.1
Salicylate	7.2±0.7	2.2±0.2	3.9±0.6	4.9±0.5	2.1±0.2
Butyrate	4.8±0.2	2.5±0.9	4.3±0.4	3.6±0.4	1.8±0.1

Figure 5.  (A) Jen1p topology based on the HHpred (http://protevo.eb.tuebingen.mpg. de/toolkit/index.php?view=hhpred), showing highlighted the location of the identified motif within TMS7. (B) Schematic representation of TMS7 (LLFAYLVVLLVGPNYLTHASQDLLPTMLRA), illustrating the position of the amino acids in the helix; the beginning of TMS7 is represented with a closed circle and the studied domain is represented in bold (C) Ribbon representation of Jen1p structure modelled to the LacY permease (HHpred Modeller), (I) View parallel to the membrane; (II) View from the cytosol side (the loop regions are not represented); (III) simplified II view with indication of the residues studied in this work. Transmembrane domains are indicated from I to XII and from N to C terminus. The colour of each transmembrane domain is maintained in all of the Figures shown in colour in Molecular Membrane Biology online. Figures were prepared with program Swiss PDB viewer (Guex & Peitsch 1997).

Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 1988; 16: 10881–10890

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Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative protein modeling. Electrophoresis 1997; 18: 2714–2723

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