The versatility of plant organic acid metabolism in leaves is underpinned by mitochondrial malate–citrate exchange

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate–citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.

ER targeting of non-imported mitochondrial carrier proteins is dependent on the GET pathway

Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins.

The Legionella pneumophila effector Lpg1137 is a homologue of mitochondrial SLC25 carrier proteins, not of known serine proteases

Many bacterial effector proteins that are delivered to host cells during infection are enzymes targeting host cell signalling. Recently, Legionella pneumophila effector Lpg1137 was experimentally characterised as a serine protease that cleaves human syntaxin 17. We present strong bioinformatic evidence that Lpg1137 is a homologue of mitochondrial carrier proteins and is not related to known serine proteases. We also discuss how this finding can be reconciled with the apparently contradictory experimental results.

Tyrosine phosphorylation by Src within the cavity of the adenine nucleotide translocase 1 regulates ADP/ATP exchange in mitochondria

Phosphorylation of adenine nucleotide translocator 1 (ANT1) at residue Y194, which is part of the aromatic ladder located within the lumen of the carrier, critically regulates mitochondrial metabolism. Recent data support the concept that members of the Src family of nonreceptor tyrosine kinases are constitutively present in mitochondria and key to regulation of mitochondrial function. Herein, we demonstrate that site mutations of ANT1 (Y190→F190, Y194→F194) mimicking dephosphorylation of the aromatic ladder resulted in loss of oxidative growth and ADP/ATP exchange activity in respiration-incompetent yeast expressing mutant chimeric yN-hANT1. ANT1 is phosphorylated at Y194 by the Src family kinase members Src and Lck, and increased phosphorylation is tightly linked to reduced cell injury in preconditioned protected vs. unprotected cardiac mitochondria. Molecular dynamics simulations find the overall structure of the phosphorylated ANT1 stable, but with an increased steric flexibility in the region of the aromatic ladder, matrix loop m2, and four helix-linking regions. Combined with an analysis of the putative cytosolic salt bridge network, we reason that the effect of phosphorylation on transport is likely due to an accelerated transition between the main two conformational states (c↔m) of the carrier during the transport cycle. Since “aromatic signatures” are typical for other mitochondrial carrier proteins with important biological functions, our results may be more general and applicable to these carriers.