Anionic Phospholipids Induce Conformational Changes in Phosphoenolpyruvate Carboxylase to Increase Sensitivity to Cathepsin Proteases

Abstract

Phosphoenolpyruvate carboxylase (PEPC) is a cytosolic, homotetrameric enzyme that serves a variety of functions in plants, acting as the primary form of CO 2 fixation in the C 4 photosynthesis pathway (C 4 -PEPC). In a previous work we have shown that C 4 -PEPC bind anionic phospholipids, resulting in PEPC inactivation. Also, we showed that PEPC can associate with membranes and to be partially proteolyzed. However, the mechanism controlling this remains unknown. Using semi purified-PEPC from sorghum leaf and a panel of PEPC-specific antibodies, we analyzed the conformational changes in PEPC induced by anionic phospholipids to cause the inactivation of the enzyme. Conformational changes observed involved the exposure of the C -terminus of PEPC from the native, active enzyme conformation. Investigation of the protease activity associated with PEPC demonstrated that cysteine proteases co-purify with the enzyme, with protease-specific substrates revealing cathepsin B and L as the major protease species present. The anionic phospholipid-induced C-terminal exposed conformation of PEPC appeared highly sensitive to the identified cathepsin protease activity and showed initial proteolysis of the enzyme beginning at the N -terminus. Taken together, these data provide the first evidence that anionic phospholipids promote not only the inactivation of the PEPC enzyme, but also its proteolysis.

Changes to the functional traits of phosphoenolpyruvate carboxylase following hybridization in C‐4 halophytes

“…Protease and phosphatases inhibitors were not added to the extraction buffer to avoid PEPC activity lost because PEPC activity in leaf extracts was measured rapidly and very diluted (See Echevarria et al ), and we performed tests and found that similar results were obtained with and without adding inhibitors (see Tables S1, S2). In addition, extracts were not desalted because our comparative essays (Table S2) and previous studies have shown similar results with and without desalting (Rodrïguez‐Penagos and Muñoz‐Clares ; Avasthi and Raghavendra ; Gandullo et al ). Anti‐body anti‐PEPC and anti‐phosphorylation site at serine close to the N‐terminal site of the enzyme were applied to confirm that during enzymatic extractions the PEPC did not lose its N‐terminal end and that crude extracts obtained rapidly in the absence of proteases inhibitors and diluted are adequate to measure PEPC activity and IC 50 values in Spartina taxa (See Fig.…”

Genetic and Pharmacological Inhibition of Autophagy Increases the Monoubiquitination of Non-Photosynthetic Phosphoenolpyruvate Carboxylase

“…It could be hypothesized that PEPC proteins directed towards selective autophagy are altered forms with a higher sensitivity to proteolytic cleavage. We have recently shown that the interaction of sorghum PEPC with anionic phospholipids changes its conformation and this increases its sensibility to cysteine proteases [ 61 ].…”

From physiology to salt marsh management challenges with sea level rise: the case of native Spartina foliosa, invasive S. densiflora and their hybrid

“…Protease and phosphatase inhibitors were not added to the extraction buffer to avoid PEPC activity lost since PEPC activity in leaf extracts was measured rapidly and very diluted (5 μl of crude extract in a final volume of 1000 μl); similar results have been obtained with and without adding inhibitors ( Echevarría et al ., 1990 ; Gallego-Tévar et al ., 2019a ). Extracts were not desalted as previous studies have shown similar results with and without desalting ( Rodríguez-Penagos and Muñoz-Clares, 1999 ; Gandullo et al ., 2019 ; Gallego-Tévar et al ., 2019a ). The total protein amount was determined following the colorimetric method of Bradford (1976 ), using bovine serum albumin as standard.…”