The fungus Neurospora crassa has provided a combination of electrophysiologic, flux, and biochemical data which strongly support what might be called the generalized chemiosmotic hypothesis of transport organization in biological membranes. New experiments proposed on the Neurospora plasma membrane are aimed in three directons: 1) to delimit or constrain the possible molecular mechanisms of transport by developing the first comprehensive kinetic model of an electrogenic ion pump (the ATP-dependent proton extrusion system) an electrogenic ion-coupled sugar transport system (the glucose/proton cotransport system); 2) to identify the cytoplasmic factors by which transport is modulated or """"""""controlled"""""""", commensurate with metabolic needs; and 3) to develop artificial membrane preparations for study of the isolated proton pump and glucose/proton cotransport system. The central technique for these experiments will be current-voltage analysis of the membrane and its individual transport systems, based on computer-controlled voltage-clamp programs. This will be supplemented by chemical and isotopic measurements of proton, sugar, and alkali cation fluxes, by ion-specific electrode determinations of intracellular pH and free calcium, and by intracellular pH-clamping via exogenous weak acids and bases. More extensive manipulation of the intracellular environment for the proton transport systems will be sought through cell perfusion; and complex metabolic events related to transport control will be monitored by nuclear magnetic resonance analysis.
