Targeting of hexokinase 1 to liver and hepatoma mitochondria

B. D. Gelb, V. Adams, S. N. Jones, L. D. Griffin, G. R. MacGregor, E. R.B. McCabe

Research output: Contribution to journalArticlepeer-review

82 Scopus citations

Abstract

The proportion of hexokinase (HK; EC 2.7.1.1) isozyme 1 (HK1) that is bound to the outer mitochondrial membrane is tissue specific and developmentally regulated. HK activity is known to be markedly elevated in many cancer cells and a significant fraction is mitochondrial bound. This study examined the role of the 15-amino acid N-terminal domain of HK1 in binding to liver and hepatoma mitochondria. A chimeric reporter construct, pCMVHKCAT, encoding this HK1 domain coupled to the chloramphenicol acetyltransferase (CAT) gene was electroporated into mouse Hepa 1-6 hepatoma cells. After digitonin treatment, cell fractions were assayed for HK, lactate dehydrogenase, and CAT activities. Digitonin (75 μg/mg of protein) caused cytosolic leak but 70% of HK remained with the pellet. HKCAT, like HK, remained predominantly with the pellet; CAT from the control, pCMVCAT, remained mostly unbound. Binding of membrane-free cell extracts to rat liver mitochondria in vitro showed 91% of the HKCAT bound, whereas only 12% of CAT bound. Specificity of HKCAT binding to mitochondria was demonstrated by competition of HK1 for HKCAT binding sites on rat liver mitochondria as well as by blockage of HKCAT binding by N,N'-dicyclohexylcarbodiimide, which covalently binds to porin and blocks HK1 binding. Deletional mutant constructs of HKCAT showed reduced binding with increasing deletion size. In summary, these studies demonstrate that the 15-amino acid N-terminal domain of HK1 is necessary and sufficient to confer mitochondrial binding properties to CAT and that there is specificity for this binding to the mitochondria.

Original languageEnglish
Pages (from-to)202-206
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number1
DOIs
StatePublished - 1992
Externally publishedYes

Keywords

  • N,N'-dicyclohexylcarbodiimide
  • chloramphenicol acetyltransferase
  • hepatoma cell line
  • reporter gene construct

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