Akio Kihara

Beclin–phosphatidylinositol 3‐kinase complex functions at the trans‐Golgi network

Autophagy is an intracellular bulk protein degradation system. Beclin is known to be involved in this process; however, its role is unclear. In this study, we showed that Beclin was co‐immunoprecipitated with phosphatidylinositol (PtdIns) 3‐kinase, which is also required for autophagy, suggesting that Beclin is a component of the PtdIns 3‐kinase complex. Quantitative analyses using a cross‐linker showed that all Beclin forms a complex with PtdIns 3‐kinase, whereas ∼50% of PtdIns 3‐kinase remains free from Beclin. Indirect immunofluorescence microscopy demonstrated that the majority of Beclin and PtdIns 3‐kinase localize to the trans‐Golgi network (TGN). Some PtdIns 3‐kinase is also distributed in the late endosome. These results suggest that Beclin and PtdIns 3‐kinase control autophagy as a complex at the TGN.

Mammalian Lass6 and its related family members regulate synthesis of specific ceramides

The Lass (longevity-assurance homologue) family members, which are highly conserved among eukaryotes, function in ceramide synthesis. In the mouse, there are at least five Lass family members, Lass1, Lass2, Lass4, Lass5 and the hitherto uncharacterized Lass6. To investigate specific roles for each Lass member in ceramide synthesis, we cloned these five mouse proteins. Overproduction of any Lass protein in cultured cells resulted in an increase in cellular ceramide, but the ceramide species produced varied. Overproduction of Lass1 increased C18:0-ceramide levels preferentially, and overproduction of Lass2 and Lass4 increased levels of longer ceramides such as C22:0- and C24:0-ceramides. Lass5 and Lass6 produced shorter ceramide species (C14:0- and C16:0-ceramides); however, their substrate preferences towards saturated/unsaturated fatty acyl-CoA differed. In addition to differences in substrate preferences, we also demonstrated by Northern blotting that Lass family members are differentially expressed among tissues. Additionally, we found that Lass proteins differ with regard to glycosylation. Of the five members, only Lass2, Lass5 and Lass6 were N-glycosylated, each at their N-terminal Asn residue. The occurrence of N-glycosylation of some Lass proteins provides topological insight, indicating that the N-termini of Lass family members probably face the luminal side of the endoplasmic reticulum membrane. Furthermore, based on a proteinase K digestion assay, we demonstrated that the C-terminus of Lass6 faces the cytosolic side of the membrane. From these data we propose topology for the conserved Lag1 motif in Lass family members, namely that the N-terminal region faces the luminal side and the C-terminal region the cytosolic side of the endoplasmic reticulum membrane.

A protease complex in the Escherichia coli plasma membrane: HflKC (HflA) forms a complex with FtsH (HflB), regulating its proteolytic activity against SecY.

Escherichia coli FtsH (HflB), a membrane‐bound ATPase is required for proteolytic degradation of uncomplexed forms of the protein translocase SecY subunit. We have now isolated SecY‐stabilizing mutations that cause an amino acid substitution in the HflK‐HflC membrane protein complex. Although HflKC protein was believed to have a proteolytic activity against lambda cII protein, deletion of hflK‐hflC did not stabilize SecY. Instead, the mutant alleles were partially dominant and overexpression of ftsH suppressed the mutational effects, suggesting that the mutant proteins antagonized the degradation of SecY. These results raise the possibility that even the wild‐type HflKC protein acts to antagonize FtsH. Consistent with this notion, the hflkC null mutation accelerated degradation of the SecY24 protein. Furthermore cross‐linking, co‐immunoprecipitation, histidine‐tagging and gel filtration experiments all indicated that FtsH and HflKC form a complex in vivo and in vitro. Finally, purified HflKC protein inhibited the SecY‐degrading activity of purified FtsH protein in vitro. These results indicate that the proteolytic activity of FtsH is modulated negatively by its association with HflKC.

Ceramide biosynthesis in keratinocyte and its role in skin function

The enucleate layer of the epidermis, i.e. the stratum corneum, is responsible for certain critical protective functions, such as epidermal permeability barrier function. Within the epidermal membrane lamella component, ceramides are the dominant lipid class by weight (over 50%) and exhibit the greatest molecular heterogeneity in terms of sphingoid base and fatty acid composition. It is now evermore important to understand how ceramide production and functions are controlled in the epidermis, since decreased epidermal ceramide content has been linked to water loss and barrier dysfunction. During the past several years, critical enzymes in ceramide biosynthesis have been identified, including ceramide synthases (CerS) and ceramide hydroxylase/desaturase. In this review, we describe the molecular heterogeneity of ceramides synthesized in the epidermis and their possible roles in epidermal permeability barrier functions. We also describe recent studies that identified the family of CerS (CerS1-CerS6) in mammals. We further focus on the roles of specific isoforms of these enzymes in synthesizing the epidermal ceramides, especially in relation to chain-length specificity. In addition, we provide experimental information, including our recent findings, as to how applying ceramide or ceramide-containing substances to skin, orally or directly, can benefit skin health.

FtsH (HflB) Is an ATP-dependent Protease Selectively Acting on SecY and Some Other Membrane Proteins

The FtsH protein is a membrane-bound ATPase of Escherichia coli that was proposed to be involved in membrane protein assembly as well as degradation of some unstable proteins. SecY, a subunit of protein translocase, is FtsH dependently degraded in vivo when it fails to associate with its partner (the SecE protein). We constructed a series of mutants in which mutations were introduced into conserved residues in the two ATP binding consensus sequences or the zinc binding sequence of FtsH. We purified wild-type and mutant FtsH proteins by making use of a polyhistidine tag attached to their carboxyl termini. Complementation analysis and ATPase activity assays in vitro indicated that, of the two sets of ATP binding sequence motifs, the one located C-terminally (A1) is essential for ATPase activity and in vivo functioning of FtsH. Wild-type FtsH protein degraded purified SecY in an ATP hydrolysis-dependent manner in vitro. Mutant proteins without ATPase activity were inactive in proteolysis. A zinc binding motif mutant showed a decreased proteolytic activity. SecY and FtsH were cross-linkable with each other in the membrane, provided that FtsH had an ATPase-inactivating mutation. These results demonstrate that FtsH binds to and degrades SecY, its A1 motif and the zinc binding motif being important for the proteolytic activity. FtsH-dependent proteolysis was also demonstrated for SecY in crude membrane extracts, whereas a majority of other membrane proteins were not degraded, indicating that FtsH has high selectivity in protein degradation.

ELOVL1 production of C24 acyl-CoAs is linked to C24 sphingolipid synthesis.

Very long-chain fatty acids (VLCFAs) exert a variety of cellular functions and are associated with numerous diseases. However, the precise pathway behind their elongation has remained elusive. Moreover, few regulatory mechanisms for VLCFAs synthesis have been identified. Elongases catalyze the first of four steps in the VLCFA elongation cycle; mammals have seven elongases (ELOVL1–7). In the present study, we determined the precise substrate specificities of all the ELOVLs by in vitro analyses. Particularly notable was the high activity exhibited by ELOVL1 toward saturated and monounsaturated C20- and C22-CoAs, and that it was essential for the production of C24 sphingolipids, which are unique in their capacity to interdigitate within the membrane as a result of their long chain length. We further established that ELOVL1 activity is regulated with the ceramide synthase CERS2, an enzyme essential for C24 sphingolipid synthesis. This regulation may ensure that the production of C24-CoA by elongation is coordinated with its utilization. Finally, knockdown of ELOVL1 caused a reduction in the activity of the Src kinase LYN, confirming that C24-sphingolipids are particularly important in membrane microdomain function.

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner.

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca2+. Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca2+-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca2+ also stimulated S1P release from ATX platelets without ATP, whereas the Ca2+-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca2+.

LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

Subunit a of proton ATPase F0 sector is a substrate of the FtsH protease in Escherichia coli.

Escherichia coli FtsH is a membrane‐bound ATPase with a proteolytic activity against the SecY subunit of protein translocase. We now report that subunit a of the membrane‐embedded F0 part of H+‐ATPase is another substrate of FtsH. Pulse‐chase experiments showed that subunit a is unstable when it alone (without F0 subunits b and c) was oversynthesized and that it is stabilized in the ftsH mutants. Selective and ATP‐dependent degradation of subunit a by purified FtsH protein was demonstrated in vitro. These results suggest that FtsH serves as a quality‐control mechanism to avoid potentially harmful accumulation of free subunit a in the membrane.

Very long-chain fatty acids: elongation, physiology and related disorders

Very long-chain fatty acids (VLCFAs) are fatty acids (FAs) with a chain-length of ≥22 carbons. Mammals have a variety of VLCFAs differing in chain-length and the number of double bonds. Each VLCFA exhibits certain functions, for example in skin barrier formation, liver homeostasis, myelin maintenance, spermatogenesis, retinal function and anti-inflammation. These functions are elicited not by free VLCFAs themselves, but through their influences as components of membrane lipids (sphingolipids and glycerophospholipids) or precursors of inflammation-resolving lipid mediators. VLCFAs are synthesized by endoplasmic reticulum membrane-embedded enzymes through a four-step cycle. The most important enzymes determining the tissue distribution of VLCFAs are FA elongases, which catalyze the first, rate-limiting step of the FA elongation cycle. Mammals have seven elongases (ELOVL1-7), each exhibiting a characteristic substrate specificity. Several inherited disorders are caused by mutations in genes involved in VLCFA synthesis or degradation. In this review, I describe the molecular mechanism of FA elongation and the responsible enzymes in mammals and yeast, as well as VLCFA-related disorders in human.