1887

Microbiology Society logo

Volume 168, Issue 3

Importance of transmembrane helix 4 of -alanine exporter AlaE in oligomer formation and substrate export activity in Free

Abstract

AlaE is the smallest amino acid exporter identified in . It exports -alanine using the proton motive force and plays a pivotal role in maintaining intracellular -alanine homeostasis by acting as a safety valve. However, our understanding of the molecular mechanisms of substrate export by AlaE is still limited because structural information is lacking. Due to its small size (149 amino acid residues), it has been speculated that AlaE functions by forming an oligomer. In this study, we performed chemical cross-linking and pull-down assays and showed that AlaE indeed generates homo-oligomers as a functional unit. Previous random mutagenesis experiments identified three loss-of-function AlaE point mutations in the predicted transmembrane helix 4 (TM4) region, two of which are present in the GxxxG motif. When alanine-scanning mutagenesis was applied to the TM4 region, the AlaE derivatives that had amino acid substitutions around the GxxxG motif showed low -alanine export activities, indicating that the GxxxG motif in TM4 plays an important role in substrate export. However, these AlaE variants with low activity could still form oligomers. We therefore concluded that AlaE forms homo-oligomers and that the GxxxG motif in the TM4 region plays an essential role in AlaE activity but is not involved in AlaE oligomer formation.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): AlaE , amino acid exporter , GxxxG motif , l-alanine and oligomerization
Funding
This study was supported by the:
  • Adaptable and Seamless Technology Transfer Program through Target-Driven R and D (Award AS232Z00075E)
    • Principle Award Recipient: HiroshiYoneyama
© 2022 The Authors
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001147
2022-03-11
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/168/3/mic001147.html?itemId=/content/journal/micro/10.1099/mic.0.001147&mimeType=html&fmt=ahah

References

  1. Kinoshita S, Udaka S, Shimono M. Studies on the amino acid fermentation part I. production of L-glutamic acid by various microorganisms. J Gen Appl Microbiol 1957; 3:193–205 [View Article]
     [Google Scholar]
  2. Vrljic M, Sahm H, Eggeling L. A new type of transporter with a new type of cellular function: L-lysine export from Corynebacterium glutamicum. Mol Microbiol 1996; 22:815–826 [View Article] [PubMed]
     [Google Scholar]
  3. Zakataeva NP, Aleshin VV, Tokmakova IL, Troshin PV, Livshits VA. The novel transmembrane Escherichia coli proteins involved in the amino acid efflux. FEBS Lett 1999; 452:228–232 [View Article] [PubMed]
     [Google Scholar]
  4. Kruse D, Krämer R, Eggeling L, Rieping M, Pfefferle W et al. Influence of threonine exporters on threonine production in Escherichia coli. Appl Microbiol Biotechnol 2002; 59:205–210 [View Article] [PubMed]
     [Google Scholar]
  5. Livshits VA, Zakataeva NP, Aleshin VV, Vitushkina MV. Identification and characterization of the new gene rhtA involved in threonine and homoserine efflux in Escherichia coli. Res Microbiol 2003; 154:123–135 [View Article] [PubMed]
     [Google Scholar]
  6. Yamada S, Awano N, Inubushi K, Maeda E, Nakamori S et al. Effect of drug transporter genes on cysteine export and overproduction in Escherichia coli. Appl Environ Microbiol 2006; 72:4735–4742 [View Article] [PubMed]
     [Google Scholar]
  7. Franke I, Resch A, Dassler T, Maier T, Böck A. YfiK from Escherichia coli promotes export of O-acetylserine and cysteine. J Bacteriol 2003; 185:1161–1166 [View Article] [PubMed]
     [Google Scholar]
  8. Pittman MS, Corker H, Wu G, Binet MB, Moir AJG et al. Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly. J Biol Chem 2002; 277:49841–49849 [View Article] [PubMed]
     [Google Scholar]
  9. Nandineni MR, Gowrishankar J. Evidence for an arginine exporter encoded by yggA (argO) that is regulated by the LysR-type transcriptional regulator ArgP in Escherichia coli. J Bacteriol 2004; 186:3539–3546 [View Article] [PubMed]
     [Google Scholar]
  10. Kutukova EA, Livshits VA, Altman IP, Ptitsyn LR, Zyiatdinov MH et al. The yeaS (leuE) gene of Escherichia coli encodes an exporter of leucine, and the Lrp protein regulates its expression. FEBS Lett 2005; 579:4629–4634 [View Article] [PubMed]
     [Google Scholar]
  11. Park JH, Lee KH, Kim TY, Lee SY. Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation. Proc Natl Acad Sci U S A 2007; 104:7797–7802 [View Article] [PubMed]
     [Google Scholar]
  12. Doroshenko V, Airich L, Vitushkina M, Kolokolova A, Livshits V et al. YddG from Escherichia coli promotes export of aromatic amino acids. FEMS Microbiol Lett 2007; 275:312–318 [View Article] [PubMed]
     [Google Scholar]
  13. Liu Q, Liang Y, Zhang Y, Shang X, Liu S et al. YjeH is a novel exporter of l-methionine and branched-chain amino acids in Escherichia coli. Appl Environ Microbiol 2015; 81:7753–7766 [View Article] [PubMed]
     [Google Scholar]
  14. Pathania A, Sardesai AA. Distinct paths for basic amino acid export in Escherichia coli: YbjE (LysO) Mediates Export of L-Lysine. J Bacteriol 2015; 197:2036–2047 [View Article] [PubMed]
     [Google Scholar]
  15. Hori H, Yoneyama H, Tobe R, Ando T, Isogai E et al. Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli. Appl Environ Microbiol 2011; 77:4027–4034 [View Article] [PubMed]
     [Google Scholar]
  16. Kim S, Ihara K, Katsube S, Hori H, Ando T et al. Characterization of the l-alanine exporter AlaE of Escherichia coli and its potential role in protecting cells from a toxic-level accumulation of l-alanine and its derivatives. Microbiologyopen 2015; 4:632–643 [View Article] [PubMed]
     [Google Scholar]
  17. Ihara K, Sato K, Hori H, Makino Y, Shigenobu S et al. Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli. J Biosci Bioeng 2017; 123:444–450 [View Article] [PubMed]
     [Google Scholar]
  18. Katsube S, Ando T, Yoneyama H. L-Alanine Exporter, AlaE, of Escherichia coli Functions as a safety valve to enhance survival under feast conditions. Int J Mol Sci 2019; 20:E4942 [View Article] [PubMed]
     [Google Scholar]
  19. Hori H, Ando T, Isogai E, Yoneyama H, Katsumata R. Identification of an L-alanine export system in Escherichia coli and isolation and characterization of export-deficient mutants. FEMS Microbiol Lett 2011; 316:83–89 [View Article] [PubMed]
     [Google Scholar]
  20. Kim S, Ihara K, Katsube S, Ando T, Isogai E et al. Impact of charged amino acid substitution in the transmembrane domain of L-alanine exporter, AlaE, of Escherichia coli on the L-alanine export. Arch Microbiol 2017; 199:105–114 [View Article] [PubMed]
     [Google Scholar]
  21. Senes A, Engel DE, DeGrado WF. Folding of helical membrane proteins: the role of polar, GxxxG-like and proline motifs. Curr Opin Struct Biol 2004; 14:465–479 [View Article] [PubMed]
     [Google Scholar]
  22. Teese MG, Langosch D. Role of GxxxG motifs in transmembrane domain interactions. Biochemistry 2015; 54:5125–5135 [View Article] [PubMed]
     [Google Scholar]
  23. Fisher R, Tuli R, Haselkorn R. A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated. Proc Natl Acad Sci U S A 1981; 78:3393–3397 [View Article] [PubMed]
     [Google Scholar]
  24. Nanatani K, Maloney PC, Abe K. Structural and functional importance of transmembrane domain 3 (TM3) in the aspartate:alanine antiporter AspT: topology and function of the residues of TM3 and oligomerization of AspT. J Bacteriol 2009; 191:2122–2132 [View Article] [PubMed]
     [Google Scholar]
  25. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685 [View Article] [PubMed]
     [Google Scholar]
  26. Rotem D, Sal-man N, Schuldiner S. In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit. J Biol Chem 2001; 276:48243–48249 [View Article] [PubMed]
     [Google Scholar]
  27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265–275 [View Article]
     [Google Scholar]
  28. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977; 74:5463–5467 [View Article] [PubMed]
     [Google Scholar]
  29. D’Este M, Alvarado-Morales M, Angelidaki I. Amino acids production focusing on fermentation technologies - A review. Biotechnol Adv 2018; 36:14–25 [View Article] [PubMed]
     [Google Scholar]
  30. Seryoung K, Yoneyama H. Amino acid exporter: a tool for the next-generation microbial fermentation. J Biotechnol Biomater 2013; 3:118
     [Google Scholar]
  31. Jones CM, Hernández Lozada NJ, Pfleger BF. Efflux systems in bacteria and their metabolic engineering applications. Appl Microbiol Biotechnol 2015; 99:9381–9393 [View Article] [PubMed]
     [Google Scholar]
  32. Wendisch VF. Metabolic engineering advances and prospects for amino acid production. Metab Eng 2020; 58:17–34 [View Article] [PubMed]
     [Google Scholar]
  33. Ubarretxena-Belandia I, Baldwin JM, Schuldiner S, Tate CG. Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J 2003; 22:6175–6181 [View Article] [PubMed]
     [Google Scholar]
  34. Tsuchiya H, Doki S, Takemoto M, Ikuta T, Higuchi T et al. Structural basis for amino acid export by DMT superfamily transporter YddG. Nature 2016; 534:417–420 [View Article] [PubMed]
     [Google Scholar]
  35. Gottschalk K-E, Soskine M, Schuldiner S, Kessler H. A structural model of EmrE, a multi-drug transporter from Escherichia coli. Biophys J 2004; 86:3335–3348 [View Article] [PubMed]
     [Google Scholar]
  36. Schushan M, Barkan Y, Haliloglu T, Ben-Tal N. C(alpha)-trace model of the transmembrane domain of human copper transporter 1, motion and functional implications. Proc Natl Acad Sci U S A 2010; 107:10908–10913 [View Article] [PubMed]
     [Google Scholar]
  37. Lisenbee CS, Miller LJ. Secretin receptor oligomers form intracellularly during maturation through receptor core domains. Biochemistry 2006; 45:8216–8226 [View Article]
     [Google Scholar]
  38. Wilson MR, Kugel S, Huang J, Wilson LJ, Wloszczynski PA et al. Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6. Biochem J 2015; 469:33–44 [View Article]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001147
Loading
Importance of transmembrane helix 4 of l-alanine exporter AlaE in oligomer formation and substrate export activity in Escherichia coli
Microbiology 168, 001147 (2022); https://doi.org/10.1099/mic.0.001147
/content/journal/micro/10.1099/mic.0.001147
/content/journal/micro/10.1099/mic.0.001147
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error