Yukio Fujiki

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Peroxisomes are organelles that are central to lipid metabolism and chemical detoxification. Despite advances in our understanding of peroxisome biogenesis, the mechanisms maintaining peroxisomal membrane proteins remain to be fully elucidated. We show here that mammalian FAF2/UBXD8, a membrane-associated cofactor of p97/VCP, maintains peroxisomal homeostasis by modulating the turnover of peroxisomal membrane proteins such as PMP70. In FAF2-deficient cells, PMP70 accumulation recruits the autophagy adaptor OPTN (Optineurin) to peroxisomes and promotes their autophagic clearance (pexophagy). Pexophagy is also induced by p97/VCP inhibition. FAF2 functions together with p97/VCP to negatively regulate pexophagy rather than as a factor for peroxisome biogenesis. Our results strongly suggest that p97/VCP^FAF2-mediated extraction of ubiquitylated peroxisomal membrane proteins (e.g., PMP70) prevents peroxisomes from inducing nonessential autophagy under steady state conditions. These findings provide insight into molecular mechanisms underlying the regulation of peroxisomal integrity by p97/VCP and its associated cofactors.

Plasmalogens are a unique family of cellular glycerophospholipids that contain a vinyl-ether bond. The synthesis of plasmalogens is initiated in peroxisomes and completed in the endoplasmic reticulum. Plasmalogens are transported to the post-Golgi compartment, including endosomes and plasma membranes, in a manner dependent on ATP, but not vesicular transport. Plasmalogens are preferentially localized in the inner leaflet of the plasma membrane in a manner dependent on P4-type ATPase ATP8B2, that associates with the CDC50 subunit. Plasmalogen biosynthesis is spatiotemporally regulated by a feedback mechanism that senses the amount of plasmalogens in the inner leaflet of the plasma membrane and controls the stability of fatty acyl-CoA reductase 1 (FAR1), the rate-limiting enzyme for plasmalogen biosynthesis. The physiological consequences of such asymmetric localization and homeostasis of plasmalogens are discussed in this review.

Phos-tag, a selective phosphate-binding molecule, and Phos-tag-based methodologies have been developed to investigate the phosphoproteome. In various analytical techniques using Phos-tag derivatives, phosphate-affinity electrophoresis using Phos-tag acrylamide, called Phos-tag SDS-PAGE, enables separation of phosphorylated proteins with a slower migration from non-phosphorylated proteins in polyacrylamide gels. The procedures for Phos-tag SDS-PAGE are largely common to those for conventional SDS-PAGE, thus being readily available for all laboratories. Phos-tag SDS-PAGE is widely applied to quantitative analysis of the overall phosphorylation state depending on the number and/or sites of the phosphate group. Phos-tag SDS-PAGE has also been introduced to the field of peroxisome study, including oxidative stress-induced and mitosis-specific phosphorylation of Pex14, a central component of the translocation machinery complex for peroxisomal matrix proteins. Here, we describe a practical protocol for Phos-tag SDS-PAGE and its application to peroxisome biogenesis research. Key words Peroxisome Phos-tag SDS-PAGE Phosphorylation Peroxins Protein import

Peroxisomes are essential organelles involved in lipid metabolisms including plasmalogen biosynthesis and β‐oxidation of very long‐chain fatty acids. Peroxisomes proliferate by the growth and division of pre‐existing peroxisomes. The peroxisomal membrane is elongated by Pex11β and then divided by the dynamin‐like GTPase, DLP1 (also known as DRP1 encoded by DNM1L gene), which also functions as a fission factor for mitochondria. Nucleoside diphosphate kinase 3 (NME3) localized in both peroxisomes and mitochondria generates GTP for DLP1 activity. Deficiencies of either of these factors induce abnormal morphology of peroxisomes and/or mitochondria, and are associated with central nervous system dysfunction. To investigate whether the impaired division of peroxisomes affects lipid metabolisms, we assessed the phospholipid composition of cells lacking each of the different division factors. In fibroblasts from the patients deficient in DLP1, NME3, or Pex11β, docosahexaenoic acid (DHA, C22:6)‐containing phospholipids were found to be decreased. Conversely, the levels of several fatty acids such as arachidonic acid (AA, C20:4) and oleic acid (C18:1) were elevated. Mouse embryonic fibroblasts from Drp1‐ and Pex11β‐knockout mice also showed a decrease in the levels of phospholipids containing DHA and AA. Collectively, these results suggest that the dynamics of organelle morphology exert marked effects on the fatty acid composition of phospholipids.

A chelator-sensitive protease in the mitochondrial matrix of the yeast, Saccharomyces cerevisiae (Biochem. Biophys. Res. Commun. 144, 277, 1987), was purified and characterized. The purified enzyme, termed protease M, specifically hydrolyzes peptide substrates on the N-side of the paired basic residues. When mastoparan was used as substrate, it cleaved Ala⁸-Leu⁹ and Lys¹¹-Lys¹² bonds as well as the N-side of Lys¹¹-Lys¹² residues. Nucleotide triphosphates stimulated the activity 3-fold at 2.5 mM. The genomic DNA sequence showed that Protease M was gene product of CYM1 known as mitochondrial presequence protease homologue in S. cerevisiae, encoding a 989-amino-acid long precursor protein. The N-terminal sequence of the purified enzyme indicated that protease M has 16-residue signal sequence and the "mature" protein consists of 973 amino acids with a molecular mass of 110 kDa. Protease M contained consensus sequence motifs of ATP-binding site very near the carboxyl terminus. The alignment of the two ATP-binding motifs is an inverted version of the common alignment. Gene-disruption of the enzyme generates mixed subunits in tetrameric MnSOD formed with 23-kDa mature and 24-kDa partial presequence-containing subunits. This report describes newly identified enzyme properties of the CYM1 gene product, protease M, and abnormal MnSOD complex formation of the disruption mutant.

Plasmalogens are a subclass of glycerophospholipid containing vinyl-ether bond at the sn-1 position of glycerol backbone. Ethanolamine-containing plasmalogens (plasmalogens) are major constituents of cellular membranes in mammalian cells and de novo synthesis of plasmalogens largely contributes to the homeostasis of plasmalogens. Plasmalogen biosynthesis is regulated by a feedback mechanism that senses the plasmalogen level in the inner leaflet of the plasma membrane and regulates the stability of fatty acyl-CoA reductase 1 (Far1), a rate-limiting enzyme for plasmalogen biosynthesis. However, the molecular mechanism underlying the localization of plasmalogens in cytoplasmic leaflet of plasma membrane remains unknown. To address this issue, we attempted to identify a potential transporter of plasmalogens from the outer to the inner leaflet of plasma membrane by focusing on phospholipid flippases, type-IV P-type adenosine triphosphatases (P4-ATPase), localized in the plasma membranes. We herein show that knockdown of ATP8B2 belonging to the class-1 P4-ATPase enhances localization of plasmalogens but not phosphatidylethanolamine in the extracellular leaflet and impairs plasmalogen-dependent degradation of Far1. Furthermore, phosphorylation of protein kinase B (AKT) is downregulated by lowering the expression of ATP8B2, which leads to suppression of cell growth. Taken together, these results suggest that enrichment of plasmalogens in the cytoplasmic leaflet of plasma membranes is mediated by ATP8B2 and this asymmetric distribution of plasmalogens is required for sensing plasmalogens as well as phosphorylation of AKT.