2.A.99 The 6TMS Ni²⁺ uptake transporter (HupE-UreJ) Family Synthesis of the hydrogen uptake (Hup) system in Rhizobium leguminosarum bv. viciae requires the function of an 18-gene cluster (hupSLCDEFGHIJK-hypABFCDEX). Among them, the hupE gene encodes a protein showing six transmembrane domains for which a potential role as a nickel permease has been proposed. Brito et al. (2010) characterized the nickel transport capacity of HupE and that of the translated product of hupE2, a hydrogenase-unlinked gene identified in the R. leguminosarum genome. HupE2 is a potential membrane protein that shows 48% amino acid sequence identity with HupE. Expression of both genes in the Escherichia coli nikABCDE mutant strain HYD723 restored hydrogenase activity and nickel transport. However, nickel transport assays revealed that HupE and HupE2 displayed different levels of nickel uptake. Site-directed mutagenesis of histidine residues in HupE revealed two motifs (HX(5)DH and FHGX[AV]HGXE) that are required for HupE functionality. An R. leguminosarum double mutant, SPF22A (hupE hupE2), exhibited reduced levels of hydrogenase activity in free-living cells, and this phenotype was complemented by nickel supplementation. Low levels of symbiotic hydrogenase activity were also observed in SPF22A bacteroid cells from lentil (Lens culinaris L.) root nodules but not in pea (Pisum sativum L.) bacteroids. Moreover, heterologous expression of the R. leguminosarum hup system in bacteroid cells of Rhizobium tropici and Mesorhizobium loti displayed reduced levels of hydrogen uptake in the absence of hupE. These data support the role of R. leguminosarum HupE as a nickel permease required for hydrogen uptake under both free-living and symbiotic conditions (Brito et al., 2010). The generalized transport reaction catalyzed by HupE is: Ni²⁺ (out) → Ni²⁺ (in)
References:
Albareda, M., A. Rodrigue, B. Brito, T. Ruiz-Argüeso, J. Imperial, M.A. Mandrand-Berthelot, and J. Palacios. (2015). Rhizobium leguminosarum HupE is a highly-specific diffusion facilitator for nickel uptake. Metallomics 7: 691-701.
Brito, B., R.I. Prieto, E. Cabrera, M.A. Mandrand-Berthelot, J. Imperial, T. Ruiz-Argüeso, and J.M. Palacios. (2010). Rhizobium leguminosarum hupE encodes a nickel transporter required for hydrogenase activity. J. Bacteriol. 192: 925-935.
Rowińska-Żyrek, M. (2017). Metal interactions with the transmembrane region of HupE Ni²⁺ transporter explain its efficiency. J Inorg Biochem 180: 33-38. [Epub: Ahead of Print]
Examples:
TC#	Name	Organismal Type	Example
2.A.99.1.1	The highly specific Ni²⁺uptake porter, HupE of 191 aas and 6 or 7 putative TMSs (Brito et al., 2010). HupE is a medium-affinity and low-capacity transporter - its periplasmic region, 22HVGLHADGTLAGLN35, binds Ni²⁺ with much higher affinity than the transmembrane 36HPFSGLDH43 one, which transports the metal into the cell (Albareda et al. 2015). The specificity of the transmembrane region is similar to that of the periplasmic one and to that of the full-length HupE (Rowińska-Żyrek 2017).	Bacteria	*HupE of Rhizobium leguminosarum* (P27650)**

2.A.99.1.2	*The 6TMS Ni²⁺ uptake porter, HupE2 (Brito et al., 2010).*	Bacteria	*HupE2 of Rhizobium leguminosarum* (Q1M8G8)**

2.A.99.1.3	Urease accessory protein, UreJ	Bacteria	*UreJ of Mesorhizobium* loti (Q98CY5)**

2.A.99.1.4	HupE/UreJ family protein of 217 aas and 7 TMSs		UreJ of Microcystis aeruginosa

2.A.99.1.5	HupE/UreJ family protein of 223 aas and 7 TMSs		HupE of Flammeovirga pacifica

2.A.99.1.6	Uncharacterized protein of 252 aas and 7 C-terminal TMSs and an N-terminal hydrophilic domain.		*UP of Niveispirillum* sp. SYP-B3756**

2.A.99.1.7	HupE/UreJ family protein of 185 aas and 7 TMSs.		HupE of Nitrincola tibetensis

2.A.99.1.8	Hydantoin utilization protein A of 214 aas and 7 TMSs.		HupE of Roseovarius litoreus