Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity

Montanini, Barbara; Blaudez, Damien; Jeandroz, Sylvain; Sanders, Dale; Chalot, Michel

doi:10.1186/1471-2164-8-107

Table 1 Mutagenesis studies on CDF members

From: Phylogenetic and functional analysis of the Cation Diffusion Facilitator (CDF) family: improved signature and prediction of substrate specificity

	Prokaryotic members						Eukaryotic member
	RmCzcD		EcZitB		EcFieF		PtdMTP1
Topological position	AA substitution	Phenotype	AA substitution	Phenotype	AA substitution	Phenotype	AA substitution	Phenotype
N ter							C30S	Partially active^e
N ter							C35S	No function^e
N ter							C64S	Partially active^e
N ter	H5R	Partially active^a
N ter	H7R	No function^a
N ter	H9R	Partially active^a
TMD I	E31D	Partially active^a	E35D	Partially active^b
TMD I			E35A	Partially active^b
TMD I	E31K	No function^a
TMD II			D50E	Partially active^a
TMD II			D50A	No function^a			D86A	No function^f
TMD II					D45C	Wild type ^c
TMD II	H49R	Not expressed ^a	H53R	No function ^a			H89K	No function ^f
TMD II	H49A	No function ^a					H89A	No function ^f
TMD II	M5OL	Hypersensitive^a	M54L	Partially active^a
TMD II					D49C	Wild type ^c
TMD II	D53E	Hypersensitive ^a
TMD II	D53A	Hypersensitive ^a	D57A	No function ^a	D49A	No function ^c	D93A	No function ^f
TMD II	D53N	Hypersensitive^a
TMDs II-III loop							G118A	No function^e
TMD IV			N135A	No function^a
TMD V					H153C	Partially active ^c
TMD V	E154D	No function ^a	H159E	No function ^a			H260D	No function ^e
TMD V	E154N	No function ^a	H159R	No function ^a
TMD V					D157C	Partially active ^d
TMD V	D158E	No function ^a	D163E	No function ^a			D264E	No function ^e
TMD V	D158A	No function ^a	D163A	No function ^a	D157A	No function ^d	D264A	No function ^e
TMD V			S167A	No function^a
			W182L	Partially active^a
TMD VI	D181A	Partially active^a	D186A	No function^a			D288A	No function^e
TMD VI	D181E	Hypersensitive^a	D186E	Partially active^a			D288E	No function^e
TMD VI	D181N	Hypersensitive^a
C ter							C291S	Wild type^e
C ter			E214A	No function^a
C ter	H237R	No function^a
C ter	H251A	Partially active^a
C ter			W245L	Partially active^a
C ter			G248T	Partially active^b
C ter							C357S	Wild type^e
C ter	H280A	No function^a
C ter	H298A	No function^a
C ter	C290S	Hypersensitive^a	C294S	Wild type^a

The results of site-directed mutagenesis analyses carried out on four CDF transporters ([14, 23, 29-31] and present study) are reported with the corresponding topological position. RmCzcD: Zn-CDF from C. metallidurans; EcZitB: Zn-CDF from E. coli; EcFieF: Fe/Zn-CDF from E. coli; PtdMTP1: Zn-CDF from P. trichocarpa × deltoides. "no function": the amino acid substitution led to the loss of any detectable function in the complementation test, comparable with the empty vector; "hypersensitive": the amino acid substitution resulted in a more sensitive phenotype, compared with the empty vector-transformed cells; "partially active": the phenotype is intermediate between the empty vector- and the wild type gene-transformed cells. EcFieF metal coordination residues [31] and the corresponding amino acids in the other transporters are in bold. References: ^a, [29]; ^b, [30]; ^c, [31]; ^d, [14]; ^e, this study; ^f, [23].

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ISSN: 1471-2164

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