Relative contribution of convergent pathways to proline is regulated by extracellular proline.

Lu1205 cells were labeled with [U-13C] glutamine (1 mM) for 8 hr in the presence of different concentrations of proline in the medium (0, 0.1, 0.3, 0.5 mM). The isotopic enrichment in glutamate and proline was calculated, and the fraction of proline derived from glutamate is indicated by the ratio of these two values. Since PYCRL is the primary enzyme for proline biosynthesis from ornithine, the fraction of proline synthesized via this route was determined from the same experiment performed in cells where PYCRL was knocked down. In this case the ornithine route is contributing unlabeled carbon to the proline pool, so knockdown of PYCRL (≥90%) reduces the amount of unlabeled carbon in the proline pool, and therefore actually increases the enrichment ratio (pro/glu) [Table S5]. Therefore the contribution of carbon from the ornithine route is calculated by determining how much unlabeled carbon must be lost (via PYCRL knockdown) to increase the enrichment ratio (pro/glu) as indicated [Table S5]. The formula for this value is:

Considering the sum of the three convergent pathways equal to 1, the contribution of proline salvage was determined as the residual from the total.