Substrate-dependent adaptive regulation and trans-inhibition of System A-mediated amino acid transport. Studies using rat hepatoma plasma membrane vesicles.

Substrate-dependent regulation of amino acid transport by System A occurs by both direct action at the carrier (trans-inhibition) and transcriptional control (adaptive regulation). While experiments with intact cells have led to working models that describe these regulatory phenomena, the use of subcellular approaches will serve to refine the present hypotheses. Adaptive induction of System A transport following amino acid starvation of cells was shown to be dependent on de novo RNA and protein synthesis, and the stimulated activity was shown to be retained in isolated plasma membrane vesicles. This stimulated transport activity was tightly associated with the plasma membrane, but could be solubilized by 4 M urea and 2.5% cholate, and recovered following reconstitution of the protein into artificial proteoliposomes. These data support the working hypothesis that adaptive induction of transport is the result of de novo synthesis and insertion into the plasma membrane of System A carrier protein. In contrast, the activity of System ASC in the vesicles from the amino acid starved cells was actually reduced by 2-5-fold when compared to amino acid-fed cells. A more rapid form of regulation of System A activity is trans-inhibition. The use of isolated plasma membrane vesicles demonstrated that trans-inhibition in whole cells did not survive membrane isolation. However, substrate loading of isolated membrane vesicles containing high levels of System A activity, produced trans-inhibition in a very specific manner in that System A substrates resulted in decreased transport activity, while those amino acids which are poor substrates for the System A carrier did not. Thus, trans-inhibition is not the result of a recycling process involving an intracellular pool of carriers, but rather can be accounted for by differences in the kinetics for amino acid binding and/or translocation on the two sides of the membrane.