Site-specific alteration of arginine 376, the unique positively charged amino acid residue in the mid-membrane-spanning regions of the proline carrier of Escherichia coli.

I Yamato,M Kotani,Y Oka,Y Anraku

doi:10.1016/s0021-9258(17)37520-8

Abstract

An alignment of 5 amino acids in the Escherichia coli proline carrier (G328-A366-L371-GR376) is common in the amino acid sequences of several Na+ symport carriers, and it has been proposed as the putative sodium binding motif (Deguchi, Y., Yamato, I., and Anraku, Y. (1990) J. Biol. Chem. 265, 21704-21708). To determine whether these amino acids are essential for Na+ symport activity as the Na+ binding site, one of the amino acids in this alignment, Arg-376, which is the only positively charged amino acid in the innermost part of the predicted membrane-spanning regions, was changed to either lysine, glutamine, or glutamic acid by oligonucleotide dependent site-specific mutagenesis. The transport and binding activities of the proline of the R376K mutant carrier were not detected at all. The activities of the other mutant carriers for uptake and binding of proline were as high as those of the wild-type carrier. These two mutant carriers were as sensitive to the proline analogue azetidine-2-carboxylate and to N-ethylmaleimide as the wild-type carrier, indicating that they have the same properties as the wild-type. The amounts of the carrier proteins expressed from these mutated putP genes were similar to that from the wild-type gene. These results imply that the Arg-376 in the proline carrier does not reside at the sodium binding site, suggesting that the similar alignment found in the amino acid sequences of several Na+ symport carriers is not essential for the transport or binding activities, although this similar alignment may have some relevance to the structure of the Na+ symporter. Furthermore, that the only Arg residue in the middle part of the predicted membrane-spanning regions is dispensable for the energy coupling activity indicates a unique difference of the coupling mechanism from the other secondary active transport systems, such as that of the lactose permease and the tetracycline/H+ antiporter.

Highlights

An alignment of 5 amino acidsin the Escherichia coli gene, the proline carrier has been amplified in cytoplasmic proline carrier (G32B-A366-L371-iGs Rc3om76m)on in the membranes [6,7]p, urified to complete homogeneity, and reconamino acid sequences of several Na' symport carriers, stituted, showing that it iosbligatorily coupled to theNa' elecand it has been proposedas the putative sodium binding trochemical gradient [5]
Tthreansport and binding activities of the proline of the R376K mutant carrier cidate the energy coupling mechanism of secondary active transport proteins, but thecrystallization of these hydrophobic membrane proteins hasbeen hampered by their instability in solution
By means of kinetic studies the Arg-376 in the proline carrier does notreside at the using theseCys + Ser mutant carriers ancdhemical modificasodium binding site, suggesting that the similar align- tions, the inactivatiomn echanism by N-ethylmaleimide ment found in the amino acid sequencesof several Na' (NEM)l has been extensively examined, suggesting that Cyssymport carriers is not essential for the transport or 344 is close to the Na' binding site [7, 20]

Summary

Introduction

An alignment of 5 amino acidsin the Escherichia coli gene, the proline carrier has been amplified in cytoplasmic proline carrier (G32B-A366-L371-iGs Rc3om76m)on in the membranes [6,7]p, urified to complete homogeneity, and reconamino acid sequences of several Na' symport carriers, stituted, showing that it iosbligatorily coupled to theNa' elecand it has been proposedas the putative sodium binding trochemical gradient [5]. The Na+-dependent transport activity of proline in intact cells and the binding activity in cytoplasmic membranes were measured using strain ST3009 harboring various putP gene mutant plasmids.

Results

Conclusion