Ligand-selective Modulation of the Permeability Transition Pore by Arginine Modification OPPOSING EFFECTS OF p-HYDROXYPHENYLGLYOXAL AND PHENYLGLYOXAL

Chemical modification of mitochondria with the arginine-specific reagents phenylglyoxal (PGO) and 2,3-butanedione (BAD) decreases the Ca(2+) sensitivity of the permeability transition pore (PTP) and stabilizes it in the closed conformation (Eriksson, O., Fontaine, E., and Bernardi, P. (1998) J. Biol. Chem. 273, 12669-12674). Unexpectedly, modification of mitochondria with the arginine-specific reagent p-hydroxyphenylglyoxal (OH-PGO) resulted instead in PTP opening. Sequential modification with OH-PGO and PGO (or BAD) revealed that the effects on the PTP depended on the order of the additions. PTP opening was observed when OH-PGO preceded, and PTP closing was observed when OH-PGO followed, the addition of PGO (or BAD). The differential effects of OH-PGO and PGO on the PTP open probability (i) were not modified by the conformation-specific ligands of the adenine nucleotide translocase bongkrekate and atractylate; and (ii) were also observed in de-energized mitochondria, indicating that the effect is exerted directly on the PTP. OH-PGO dramatically sensitized PTP opening, which was triggered by depolarization even in the presence of EGTA. These data show that arginine modification modulates the PTP conformation in a ligand-selective fashion and suggest that the effects of OH-PGO, PGO, and BAD are mediated by the same arginine residues. We analyzed the structure of the arginine adducts by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry using a test peptide and N-acetylarginine. The results indicate that both OH-PGO and PGO react with arginine at a stoichiometry of 2:1 and form stable adducts that may be feasible to identify the PTP at the molecular level

LACTB is a filament-forming protein localized in mitochondria

LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function

Rab8-dependent Recycling Promotes Endosomal Cholesterol Removal in Normal and Sphingolipidosis Cells

The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann–Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits