Binding of rat brain hexokinase to recombinant yeast mitochondria: identification of necessary molecular determinants.
The association in vitro of rat brain hexokinase to mitochondria from rat liver or yeast (wild type, porinless, or expressing recombinant human porin) was studied in an effort to identify minimal requirements for each component. A short hydrophobic N-terminal peptide of hexokinase, readily cleavable by proteases, is absolutely required for its binding to all mitochondria. Mammalian porins are significantly cleaved at two positions in putative cytoplasmic loops around residues 110 and 200, as determined by proteolytic-fragment identification using antibodies. Recombinant human porin in yeast mitochondria is more sensitive to proteolysis than wild-type porin in rat liver mitochondria. Recombinant yeast mitochondria, harboring several natural or engineered porins from various sources, bind hexokinase to variable extent with marked preference for the mammalian porin1 isoform. Genetic alteration of this isoform at the C-, but not the N-terminal, results in a significant reduction of hexokinase binding ability. Macromolecular crowding (dextran) promotes a stronger association of the enzyme to all recombinant mitochondria, as well as to proteolytically digested organelles. Consequently, brain hexokinase association with heterologous mitochondria (yeast) in these conditions occurs to an extent comparable to that with homologous (rat) mitochondria. The study, also pertinent to the topology and organization of porin in the membrane, represents a necessary first step in the functional investigation of the physiological role of mammalian hexokinase binding to mitochondria in reconstituted heterologous recombinant systems, as models to cellular metabolism.
Azoulay-Zohar
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Department of Life Sciences, Ben Gurion University of the Negev, Beer Sheva, Israel. aflaloc@bgumail.bgu.ac.il
Aflalo
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D000818:Animals; D001921:Brain / Q000201:enzymology*; D002384:Catalysis / Q000187:drug effects; D003911:Dextrans / Q000494:pharmacology; D006593:Hexokinase / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D018272:Porins / Q000378:metabolism / Q000494:pharmacology; D015202:Protein Engineering; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*; D014908:Triticum / Q000737:chemistry
D003911:Dextrans; D018272:Porins; D011956:Receptors, Cell Surface; D006593:Hexokinase; D010447:Peptide Hydrolases