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Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone

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Abstract

Molecular chaperones facilitate the correct folding of other proteins, and heat shock proteins form one of the major classes of molecular chaperones. Heat shock protein 70 (Hsp70) has been extensively studied, and shown to be critically important for cellular protein homeostasis in almost all prokaryotic and eukaryotic systems studied to date. Since there have been very limited studies conducted on coelacanth chaperones, the main objective of this study was to genetically and biochemically characterize a coelacanth Hsp70. We have successfully isolated an Indonesian coelacanth (L. menadoensis) hsp70 gene, Lmhsp70, and found that it contained an intronless coding region and a potential upstream regulatory region. Lmhsp70 encoded a typical Hsp70 based on conserved structural and functional features, and the predicted upstream regulatory region was found to contain six potential promoter elements, and three potential heat shock elements (HSEs). The intronless nature of the coding region and the presence of HSEs suggested that Lmhsp70 was stress-inducible. Phylogenetic analyses provided further evidence that Lmhsp70 was probably inducible, and that it branched as a clade intermediate between bony fish and tetrapods. Recombinant LmHsp70 was successfully overproduced, purified and found to be functional using ATPase activity assays. Taken together, these data provide evidence for the first time that the coelacanth encodes a functional molecular chaperone system.

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Abbreviations

ADP:

Adenosine 5′-diphosphate

ATP:

Adenosine 5′-triphosphate

BAC:

Bacterial artificial chromosome

HSE:

Heat shock element

Hsp70:

Heat shock protein 70

IPTG:

Isopropyl-1-thio-β-d-galactopyranoside

kDa:

Kilodalton

LcHsp70:

Latimeria chalumnae Hsp70

LmHsp70:

Latimeria menadoensis Hsp70

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgments

This research was funded by the South African Department of Science and Technology [administered through South African Institute of Aquatic Biodiversity (SAIAB)]. We are grateful to the German Student Exchange Services (DAAD) and the National Research Foundation (NRF) for study scholarships to KWM. MJ was funded by a study scholarship from the Andrew Mellon Foundation, and E-RP was awarded a Claude Leon Foundation postdoctoral fellowship. Research support for JR is from the National Institutes of Health (R01-CA-108982-02). The authors gratefully acknowledge Mr J. Danke for technical assistance with the L. menadoensis BAC library screening and Dr Jacquie Schein for assistance with the BAC fingerprinting analysis.

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Authors and Affiliations

  1. Biomedical Biotechnology Research Unit, Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, Grahamstown, 6410, South Africa

    Keoagile W. Modisakeng, Meesbah Jiwaji, Eva-Rachele Pesce, Rosemary A. Dorrington & Gregory L. Blatch

  2. Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY, 14642, USA

    Jacques Robert

  3. Benaroya Research Institute at Virginia Mason, Seattle, WA, 98101, USA

    Chris T. Amemiya

  4. Department of Biology, University of Washington, Seattle, WA, 98195, USA

    Chris T. Amemiya

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  1. Keoagile W. Modisakeng
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  2. Meesbah Jiwaji
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  3. Eva-Rachele Pesce
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  4. Jacques Robert
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  5. Chris T. Amemiya
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  7. Gregory L. Blatch
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Correspondence to Gregory L. Blatch.

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Communicated by M. Hammerschmidt.

K. W. Modisakeng and M. Jiwaji contributed equally to this study.

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Modisakeng, K.W., Jiwaji, M., Pesce, ER. et al. Isolation of a Latimeria menadoensis heat shock protein 70 (Lmhsp70) that has all the features of an inducible gene and encodes a functional molecular chaperone. Mol Genet Genomics 282, 185–196 (2009). https://doi.org/10.1007/s00438-009-0456-4

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