COMPLEX SUBCELLULAR-DISTRIBUTION OF SODIUM-DEPENDENT AMINO-ACID-TRANSPORT SYSTEMS IN KIDNEY CORTEX AND LLC-PK1 CL4 CELLS/

To characterize the amino acid transport system in basalateral membran es and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activit y in rabbit proximal tubular cells and LLC-PK1/Cl4 cells. A three-dime nsional separation procedure based on differential sedimentation, dens ity gradient centrifugation, and counter-cur-rent distribution resolve d 21 physically and biochemically distinct membrane populations from r abbit cortex. Inhibition of L-alanine transport by phenylalanine and N -(methylamino)isobutyric acid was used to delineate parallel amino aci d transport pathways. Population n was identified as brush border memb ranes by virtue of its 16-fold maltase enrichment; 94% of its Na+depen dent alanine transport activity was mediated by systems previously sho wn to be characteristic of brush border membranes. Two populations, c' and c'', which accounted for 25% of the total Na,K-ATPase activity, w ere identified as basalateral membranes on the basis of Na,K-ATPase cu mulative enrichment factors of 15 and 21; 82% of the total alanine tra nsport in these populations was mediated by a Na+-independent system s imilar to the classical system L. Na,K-ATPase, Na+-independent and Na-dependent alanine transport activities were associated with intracell ular membrane populations as well as with the plasma membranes. The ma jor intracellular locus of Na,K-ATPase activity, population i accounte d for roughly 31% of the Na, K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population l, which was identified as endoplasmic reticulum because it was the majo r locus of membrane-bound NADPH cytochrome c reductase activity, conta ined 44% of the total system A transport. Three distinct Golgi-derived populations, m', m'', and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transp ort was present in membranes of intracellular and possibly apical orig in. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.