Nucleotide and DNA uptake by Neurospora crassa: involvement of an uptake stimulating protein

Abstract

The basal and DUSF (DNA-uptake-stimulating factor, described previously by Schablik and Szabó (1981) FEMS Microbiol. Lett. 10, 395-397) stimulated uptake of [3H]DNA and radioactive nucleotides by Neurospora crassa (FGSC 1118, slime) cell-wall-less strain was studied. The uptake of [3H]DNA by the cells is a saturable and time-dependent process. The pH and temperature optimum for [3H]DNA uptake are pH 7 and 27 degrees C, respectively. Both basal and DUSF-stimulated uptake of [3H]DNA are inhibited by 2,4-dinitrophenol and iodoacetic acid. The inhibition of the basal uptake of [3H]DNA by cycloheximide is greater than the DUSF-stimulated uptake. The DUSF enhanced not only DNA-uptake but also that of the oligonucleotides and mononucleotides. DUSF binds both macromolecular [3H]DNA and [14C]AMP, and there might be competition between nucleotides and DNA for the binding to DUSF. Polyclonal antibodies prepared against DUSF inhibited both basal and DUSF-enhanced [3H]DNA uptake. DUSF was detected by immunoblotting among the proteins isolated from purified N. crassa cell-membranes. DUSF might be a receptor protein for DNA and nucleotides in the cell-membrane of N. crassa and play a role in DNA and nucleotide uptake.