Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Kyohei Higashi,Yoshiharu Sakamaki,Emiko Herai,Risa Demizu,Takeshi Uemura,Sunil D Saroj,Risa Zenda,Yusuke Terui,Kazuhiro Nishimura,Toshihiko Toida,Keiko Kashiwagi,Kazuei Igarashi

doi:10.1074/jbc.m110.186536

Kyohei Higashi, Yoshiharu Sakamaki + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m110.186536

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Abstract

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Highlights

That both PotE and CadB play important roles for cell growth at acidic pH [9]
Identification of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake—To determine which amino acids in PotB and PotC are involved in spermidine uptake, PotB and PotC mutants that influence spermidine uptake were isolated by random mutagenesis
E. coli MA261 potB::Km was transformed with the mutant potABCD, and spermidine uptake was measured

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains, Plasmids, and Culture Conditions—A polyamine-requiring mutant, E. coli MA261 Western Blot Analysis of PotA, B, C, and D Proteins on Rightside-out Membrane Vesicles—Antibodies against PotA and PotD were prepared as described previously [32]. Assay for PotD Binding to Rightside-out Membrane Vesicles— The reaction mixture (95 ␮l), containing buffer 1 (50 mM potassium phosphate buffer, pH 6.6, 50 mM Hepes-KOH, pH 7.6, 10 mM MgSO4, 20 mM ascorbic acid, and 10 ␮M phenazine methosulfate), 50 ␮g of protein of rightside-out membrane vesicles prepared from E. coli DR112/pACYCpotABC [22], and 1 ␮g of PotD, was preincubated at 30 °C for 5 min. In Vitro Translation of PotB, PotC, and PotD Proteins— DNA-dependent transcription-translation reactions [41] were carried out as described previously [42] with some modifications using an E. coli T7 S30 Extract System for Circular DNA (Promega). Radioactivity of labeled protein was quantified using a BAS-2000II imaging analyzer (Fuji Film)

RESULTS

DISCUSSION

Kashiwagi and Kazuei Igarashi