FUNCTIONS OF POTA AND POTD PROTEINS IN SPERMIDINE-PREFERENTIAL UPTAKESYSTEM IN ESCHERICHIA-COLI

Functions of potA and -D proteins in the spermidine-preferential uptak e system, which consists of potA, -B, -C, and -D proteins, were studie d. Spermidine uptake activity was lost when the gene for potA or potD protein was disrupted, and transformation of the cells with potA or po tD gene recovered the uptake activity. PotD protein was found to bind spermidine with a 3.2 muM dissociation constant. Spermidine uptake by membrane vesicles prepared from Escherichia coli DR112 containing the genes for potA, -B, and -C proteins was strongly dependent on the addi tion of potD protein, and its optimal concentration was 5 muM when 10 muM spermidine was used as substrate. The ATP dependence of spermidine uptake was examined with the atp mutant of E. coli. The uptake was co mpletely dependent on ATP. When the membrane potential was extinguishe d by carbonyl cyanide m-chlorophenylhydrazone, the uptake activity was decreased by 60% even if ATP existed. This suggests that the membrane potential is also involved in the spermidine uptake. ATP was found to bind to potA protein. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of potA protein, which is located between two consensus amino acid sequences for nucleotide binding, was replaced b y methionine. Although the amount of mutated potA protein expressed in E. coli cells was the same as that of normal potA protein and the mut ated protein was membrane-associated, no significant spermidine uptake was observed. The results taken together indicate that potA and -D pr oteins are absolutely necessary for spermidine uptake in conjunction w ith the two channel forming proteins (potB and -C).