Subcellular Localization of Proline Metabolic Enzymes in Lung Adenocarcinoma

Background: Proline is a nonessential amino acid and its metabolism is increasingly recognized as a key regulator of mitochondrial function, cellular redox balance, and stress response with important roles in several diseases, particularly cancer. Proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate reductase (PYCR) enzymes are central to proline metabolism, where two PRODH isoforms—PRODH1 and PRODH2—catalyze the first step of proline and hydroxyproline catabolism, respectively, and three PYCR isoforms—PYCR1, PYCR2, and PYCR3—catalyze the last step of proline synthesis. The subcellular localization of these enzymes remains incompletely defined. Methods: We selected a lung adenocarcinoma cell line to extensively characterize the subcellular localization of proline metabolic enzymes by cellular fractionation, carbonate extraction, proteinase-K digestion assays, and immunofluorescence microscopy. Results and Conclusions: Our findings show that PYCR1 and PYCR2 localize to the mitochondrial matrix whereas PYCR3 localizes to the cytoplasm. Catabolic enzymes PRODH1 and PRODH2 localize to the inner mitochondrial membrane with PRODH2 exposed to the inner membrane space. These findings provide a framework for investigating proline metabolism in cancer.