IDENTIFICATION OF THE REGION OF A 14-KILODALTON PROTEIN OF RHODOCOCCUS RUBER THAT IS RESPONSIBLE FOR THE BINDING OF THIS PHASIN TO POLYHYDROXYALKANOIC ACID GRANULES

The function of the polyhydroxyalkanoic acid (PHA) granule-associated GA14 protein of Rhodococcus ruber was investigated in Escherichia coli XL1-Blue, which coexpressed this protein with the polyhydroxybutyric acid (PHB) biosynthesis operon of Alcaligenes eutrophus. The GA14 prot ein had no influence on the biosynthesis rate of PHB in E. coli XL1-Bl ue(pSKCO7), but this recombinant E. coli strain formed smaller PHB gra nules than were formed by an E. coli strain that expressed only the PH B operon. Immunoelectron microscopy with GA14-specific antibodies demo nstrated the binding of GA14 protein to these mini granules. In a prev ious study, two hydrophobic domains close to the C terminus of the GA1 4 protein were analyzed, and a working hypothesis that suggested an an choring of the GA14 protein in the phospholipid monolayer surrounding the PHA granule core by these hydrophobic domains was developed (U. Pi eper-Furst, M. H. Madkour, F. Mayer, and A. Steinbuchel, J. Bacteriol. 176:4328-4337 1994). This hypothesis was confirmed by the constructio n of C-terminally truncated variants of the GA14 protein Lacking the s econd or both hydrophobic domains and by the demonstration of their in ability to bind to PHB granules. Further confirmation of the hypothesi s was obtained by the construction of a fusion protein composed of the acetaldehyde dehydrogenase II of A. eutrophus and the C terminus of t he GA14 protein containing both hydrophobic domains and by its affinit y to native and artificial PHB granules.