ANALYSIS OF A 24-KILODALTON PROTEIN ASSOCIATED WITH THE POLYHYDROXYALKANOIC ACID GRANULES IN ALCALIGENES-EUTROPHUS

A 5.0-kbp genomic EcoRI restriction fragment which complemented a thir d subclass of polyhydroxyalkanoic acid (PHA)-leaky mutants of A, eutro phus that accumulated PHA at a lower rate than the wild type was clone d from Alcaligenes eutrophus H16, A 687-bp phaP(Ae) gene on this fragm ent encoded a 24-kDa protein (M(r) = 23,963), which was referred to as the GA24 protein, The GA24 protein was solubilized from the granules and purified to electrophoretic homogeneity, and antibodies against th e GA24 protein were obtained, The GA24 protein bound to the surface of PHA granules, as revealed by immunoelectron microscopy of whole cells and of artificial PHA granules. The GA24 protein contributed approxim ately 5% (wt/wt) of the total cellular protein, and it was the predomi nant protein present in the granules, It was synthesized only in cells accumulating PHA and only in amounts that could be bound to the granu les; no soluble GA24 protein was detected, Tn5::mob-induced phaP(Ae) m utants which were unable to synthesize intact GA24 protein formed only one large PHA granule per cell. The amino acid sequence of the GA24 p rotein revealed two closely related stretches consisting exclusively o f nonhydrophilic amino acids at the C-terminal region, which are presu mably involved in the binding of GA24 to the granules, as was recently proposed for a similar protein in Rhodococcus ruber, The GA24 protein seems to be a representative of phasins, which are a new class of pro tein that form a layer at the surface of PHA granules, like oleosins, which form a layer at the surface of triacylglycerol inclusions in oil seed plants.