Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.     

Phosphatidylinositol-4-Phosphate 5-Kinases and Phosphatidylinositol 4,5-Bisphosphate Synthesis in the Brain

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIα, PIPKIβ, and PIPKIγ. PIPKIγ has been shown to play a role in PI(4,5)P₂ synthesis in brain, and the absence of PIPKIγ is incompatible with postnatal life. Conversely, mice lacking PIPKIα or PIPKIβ (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIα and PIPKIβ to PI(4,5)P₂ synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIγ, in the absence of both PIPKIα and PIPKIβ, can support life to adulthood. In addition, PIPKIα alone, but not PIPKIβ alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIα and PIPKIγ during embryogenesis. This is consistent with early embryonic expression of PIPKIα and PIPKIγ but not of PIPKIβ. PIPKIβ expression in brain correlates with neuronal differentiation. The absence of PIPKIβ does not impact embryonic development in the PIPKIγ knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIγ KO (death occurs within minutes rather than hours). Analysis of PIP₂ in brain reveals that only the absence of PIPKIγ significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIγ in mammals and imply that PIPKIα and PIPKIβ function in the generation of specific PI(4,5)P₂ pools that, at least in brain, do not have a major impact on overall PI(4,5)P₂ levels.     

Differential Roles of C-terminal Eps15 Homology Domain Proteins as Vesiculators and Tubulators of Recycling Endosomes

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.     

The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.The early work on retromer revealed its role in the trafficking of cargo proteins between endosomes and the trans-Golgi network (TGN),² although recently, retromer involvement in many other physiological and developmental processes has been uncovered (1, 2). The best studied proteins associated with retromer activity are intracellular sorting receptors such as the yeast vacuolar protein-10 (Vps10) and mammalian mannose 6-phosphate receptors (3, 4). These receptors sort acid hydrolases, enzymes essential for protein degradation, out of the TGN into the yeast vacuole or the mammalian lysosome. Upon releasing their substrates, these cargos traffic back to the TGN to mediate further rounds of cargo-hydrolase transportation. Similar retrograde trafficking of cargo proteins involving signaling molecules such as Wnt and amyloid precursor protein (APP) are thought to be critical for their secretion and function (5, 6). Retrograde transportation is highly regulated by the heteropentameric retromer complex that consists of a sorting nexin (SNX) dimer (e.g. Vps5 and Vps17 in yeast) and a Vps26/29/35 trimer (7). In mammals, the binding of the SNX dimer to specific phosphatidylinositol (PtdIns) determines its subcellular membrane association and governs the recruitment of the Vps trimer to endosomal compartments. Mammalian orthologs of the trimer have been biochemically characterized, and their interaction and function in cargo protein trafficking is well established (8). More recently, crystal structures of three Vps proteins in the trimer suggested how this trimer interacts with the SNX dimer and cargo proteins as well as with curved membranes (9–12). In the SNX dimer, SNX1 and SNX2 are thought to be interchangeable Vps5 orthologs (13, 14). The NMR structure of SNX1 revealed details of PI(3)P specific binding, thereby explaining its role in endosomal trafficking (15). The identity for SNX5 as a potential functional mammalian ortholog of Vps17, however, was not revealed until recently.Although initially identified as a Fanconi anemia complementation group A (FANCA)-binding protein (16), SNX5 was later shown to play an important role in membrane trafficking (17–19). SNX5 contains a PX domain (SNX5-PX) that is the signature feature in defining the SNX family, composed of 30 members at present (20) (Fig. 1B). In addition, SNX5 possesses a C-terminal BAR (Bin/Amphiphysin/Rvs) domain that has been reported to interact with a number of other proteins involved in endosomal trafficking (17, 21–27). It functions as a dimerization module that senses and/or induces membrane curvature (28, 29). Our previous biochemical study suggested a specific interaction between SNX5 and SNX1 through which the two SNXs mutually influence each other''s effect in endosomal trafficking of epidermal growth factor receptor upon epidermal growth factor stimulation (17). In support of this observation are several recent reports that indicate a critical role of SNX5 and the closely related SNX6, beyond that of SNX1 and SNX2, on retrograde sorting of mannose 6-phosphate receptor (24, 27). Therefore, SNX5 and SNX6 may be functionally interchangeable orthologs of Vps17 in mammalian cells (7, 24). Furthermore, in contrast to some reports (18, 30), SNX5 partially localizes to late endosomes and the TGN, exhibiting very low binding affinity for PtdIns(3)P (17), the substrate for phox domain proteins associating with early endosome association. Therefore, the subcellular localization and function of the SNX dimer in SNX5 function may depend on its unique structure that is different from other known PX domains.Open in a separate windowFIGURE 1.Amino Acid sequence alignment of phox domains and domain architecture of the mammalian sorting nexin family. A, comparative sequence alignment of PX domains for residues equivalent to Gly⁴⁹–Leu¹¹⁹ of the p40-PX domain (adapted from Worby and Dixon (21)). Prolines in the Pro-X-X-Pro motif are highlighted in yellow, and residues involved in phospholipid binding in the p40-PX domain are boxed in magenta. Arg⁵⁸ and Arg¹⁰⁵ are marked with magenta triangles, and Tyr⁵⁹ and Lys⁹² are marked with black stars at the bottom of the sequences. The two conserved Arg residues and Lys⁹² of p40-PX in other PX domains are highlighted in dark blue boxes; those corresponding to Tyr⁵⁹ are boxed in green. The secondary structure elements of p40-PX are indicated by yellow arrows (β-sheets) and red ovals (α-helices). The three sequence stretches that are unique in SNX5-PX (or SNX6-PX) are enclosed in a bright blue box. B, domain architecture of SNX family members. The four classes within the SNX family are designated as PX SNXs, PX-BAR SNXs, SH3-PX-BAR, and PX-other domain SNXs. Each individual domain is depicted in a different color and/or shape. The following domains are depicted: PX (phox), BAR (Bin-Amphiphysin-Rvs), SH3 (Src homology 3), TM (transmembrane), PXA (PX domain-associated), RGS (regulator of G-protein signaling), MIT (microtubule interacting and trafficking), B41 (band 4.1 homology), TPR (tetratricopeptide repeat), PDZ (postsynaptic protein PSD-95/SAP90, the Drosophila melanogaster septate junction protein Discs-large, and the tight junction protein ZO-1), and RA (Ras association).Most PX domains of SNX family proteins preferentially bind PtdIns(3)P (30–34), with few exceptions that interact with other PtdIns (30, 32, 35). There are about a dozen structurally characterized PX domains from the SNX family or other PX domain-containing proteins currently deposited in the Protein Data Bank (PDB) data base. Their structures all share common core features, a three-stranded β-sheet that is abutted by three α-helices and an irregular strand containing the PXXP region. Analyses of the representative p47-PX and SNX3-PX domain structures suggested that PtdIns(3)P binding involves two conserved Arg residues at positions equivalent to Arg⁵⁸ and Arg¹⁰⁵ in p40-PX (36). Because equivalent Arg residues are found in the PX domains of most SNX family members, it is generally assumed that all SNX proteins interact with the PtdIns(3)P-enriched elements of the early endocytic compartments. The amino acid sequences of the PX domains of both SNX5 and SNX6, however, lack the two conserved Arg residues that are involved in PtdIns(3)P binding as well as comprising a ∼30-residue insertion immediately after the PXXP motif (Fig. 1A). In addition, the PXXP motif is extended into a double PXXP motif with the sequence PXXPXXP. These unique sequence features set SNX5/6 apart from the other SNX family members. In the p40-PX domain and yeast SNX3, the two conserved Arg residues, the loop between the PXXP motif, and the α3-helix are involved in forming the binding pocket for the phosphate groups of PtdIns(3)P (36, 37). Therefore, changes in length and sequence in this region in SNX5/6-PX are expected to have profound impact on the specific structure and conformation required for PtdIns recognition.To elucidate how its unique sequence features influence the function of SNX5 in retromer-mediated retrograde membrane trafficking, we structurally investigated the SNX5-PX domain by NMR spectroscopy and x-ray crystallography. Using direct NMR titrations, we established the PtdIns binding specificity of SNX5-PX. The high resolution (1.5 Å) crystal structure of the domain revealed its distinct features when compared with previously known family members. Our results demonstrate that the SNX5-PX domain is indeed unique, both with respect to its structure as well as with respect to ligand binding. These findings have important implications for the function of SNX5 in the subcellular membrane trafficking and retrograde sorting.     

A ubiquitin-interacting motif (UIM) is essential for Eps15 and Eps15R ubiquitination

An important negative control mechanism in the signaling of epidermal growth factor (EGF) is the endocytosis and subsequent degradation of activated EGF receptors. Eps15 and its related partner Eps15R play a key role in clathrin-mediated endocytosis of transmembrane receptors. Upon EGF stimulation of the cell, Eps15 becomes both phosphorylated on tyrosine residues and monoubiquitinated. Although tyrosine phosphorylation of Eps15 has been implicated in EGF receptor internalization, the function of Eps15 ubiquitination is not known. Using a mutational approach, we have found that the second ubiquitin-interacting motif (UIM) of Eps15 and Eps15R is essential for their ubiquitination. This UIM partially overlaps with the recently characterized nuclear export signal in Eps15. We show that these two overlapping motifs have different structural requirements with respect to nuclear export signal versus ubiquitination signal activity. Our data demonstrate that the UIM does not contain the ubiquitin acceptor site but functions as a recruitment site for the ubiquitination machinery leading to the monoubiquitination of both Eps15 and Eps15R.     

The Structure of Myristoylated Mason-Pfizer Monkey Virus Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Its Membrane Binding

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C₈ fatty acid chains was monitored by observation of concentration‐dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in ³¹P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein–phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a ¹³C-filtered/¹³C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P₂ binding was not strong enough for triggering of the myristoyl‐switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.     

HIV-1 Suppressive Sequences Are Modulated by Rev Transport of Unspliced RNA and Are Required for Efficient HIV-1 Production

The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production.     

Depolarization Increases Phosphatidylinositol (PI) 4,5-Bisphosphate Level and KCNQ Currents through PI 4-Kinase Mechanisms

A growing body of evidence shows that membrane phosphatidylinositol 4,5-bisphosphates (PtdIns(4,5)P², PIP²) play an important role in cell signaling. The presence of PIP² is fundamentally important for maintaining the functions of a large number of ion channels and transporters, and for other cell processes such as vesicle trafficking, mobility, and endo- and exocytosis. PIP² levels in the membrane are dynamically modulated, which is an important signaling mechanism for modulation of PIP²-dependent cellular processes. In this study, we describe a novel mechanism of membrane PIP² modulation. Membrane depolarization induces an elevation in membrane PIP², and subsequently increases functions of PIP²-sensitive KCNQ potassium channels expressed in Xenopus oocytes. Further evidence suggests that the depolarization-induced elevation of membrane PIP² occurs through increased activity of PI4 kinase. With increased recognition of the importance of PIP² in cell function, the effect of membrane depolarization in PIP² metabolism is destined to have important physiological implications.     

ORP2 Delivers Cholesterol to the Plasma Membrane in Exchange for Phosphatidylinositol 4, 5-Bisphosphate (PI(4,5)P2)

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A Triple-Arginine Motif in the Amino-Terminal Domain and Oligomerization Are Required for HIV-1 Inhibition by Human MX2

We have employed molecular genetic approaches to understand the domain organization of the HIV-1 resistance factor myxovirus resistance 2 (MX2). First, we describe an essential triple-arginine motif in the amino-terminal domain. Second, we demonstrate that this 91-residue domain mediates antiviral activity when appended to heterologous proteins, and we provide genetic evidence that protein oligomerization is required for MX2 function. These insights will facilitate future work aiming to elucidate MX2''s mechanism of action.     

Uncleaved ApoM Signal Peptide Is Required for Formation of Large ApoM/Sphingosine 1-Phosphate (S1P)-enriched HDL Particles

Apolipoprotein M (apoM), a plasma sphingosine 1-phosphate (S1P) carrier, associates with plasma HDL via its uncleaved signal peptide. Hepatocyte-specific apoM overexpression in mice stimulates formation of both larger nascent HDL in hepatocytes and larger mature apoM/S1P-enriched HDL particles in plasma by enhancing hepatic S1P synthesis and secretion. Mutagenesis of apoM glutamine 22 to alanine (apoM^Q22A) introduces a functional signal peptidase cleavage site. Expression of apoM^Q22A in ABCA1-expressing HEK293 cells resulted in the formation of smaller nascent HDL particles compared with wild type apoM (apoM^WT). When apoM^Q22A was expressed in vivo, using recombinant adenoviruses, smaller plasma HDL particles and decreased plasma S1P and apoM were observed relative to expression of apoM^WT. Hepatocytes isolated from both apoM^WT- and apoM^Q22A-expressing mice displayed an equivalent increase in cellular levels of S1P, relative to LacZ controls; however, relative to apoM^WT, apoM^Q22A hepatocytes displayed more rapid apoM and S1P secretion but minimal apoM^Q22A bound to nascent lipoproteins. Pharmacologic inhibition of ceramide synthesis increased cellular sphingosine and S1P but not medium S1P in both apoM^WT and apoM^Q22A hepatocytes. We conclude that apoM secretion is rate-limiting for hepatocyte S1P secretion and that its uncleaved signal peptide delays apoM trafficking out of the cell, promoting formation of larger nascent apoM- and S1P-enriched HDL particles that are probably precursors of larger apoM/S1P-enriched plasma HDL.     

Rbm15-Mkl1 Interacts with the Setd1b Histone H3-Lys4 Methyltransferase via a SPOC Domain That Is Required for Cytokine-Independent Proliferation

The Rbm15-Mkl1 fusion protein is associated with acute megakaryoblastic leukemia (AMKL), although little is known regarding the molecular mechanism(s) whereby this fusion protein contributes to leukemogenesis. Here, we show that both Rbm15 and the leukemogenic Rbm15-Mkl1 fusion protein interact with the Setd1b histone H3-Lys4 methyltransferase (also known as KMT2G). This interaction is direct and requires the Rbm15 SPOC domain and the Setd1b LSD motif. Over-expression of Rbm15-Mkl1 in the 6133 megakaryoblastic leukemia cell line, previously established by expression of the Rbm15-Mkl1 fusion protein in mice (Mercher et al., [2009] J. Clin. Invest. 119, 852-864), leads to decreased levels of endogenous Rbm15 and increased levels of endogenous Mkl1. These cells exhibit enhanced proliferation and cytokine-independent cell growth, which requires an intact Rbm15 SPOC domain that mediates interaction between the Rbm15-Mkl1 fusion protein and the Setd1b methyltransferase. These results reveal altered Setd1b complex function and consequent altered epigenetic regulation as a possible molecular mechanism that mediates the leukemogenic activity of the Rbm15-Mkl1 fusion protein in AMKL.     

FAH Domain Containing Protein 1 (FAHD-1) Is Required for Mitochondrial Function and Locomotion Activity in C. elegans

The fumarylacetoacetate hydrolase (FAH) protein superfamily of metabolic enzymes comprises a diverse set of enzymatic functions, including ß-diketone hydrolases, decarboxylases, and isomerases. Of note, the FAH superfamily includes many prokaryotic members with very distinct functions that lack homologs in eukaryotes. A prokaryotic member of the FAH superfamily, referred to as Cg1458, was shown to encode a soluble oxaloacetate decarboxylase (ODx). Based on sequence homologies to Cg1458, we recently identified human FAH domain containing protein-1 (FAHD1) as the first eukaryotic oxaloacetate decarboxylase. The physiological functions of ODx in eukaryotes remain unclear. Here we have probed the function of fahd-1, the nematode homolog of FAHD1, in the context of an intact organism. We found that mutation of fahd-1 resulted in reduced brood size, a deregulation of the egg laying process and a severe locomotion deficit, characterized by a reduced frequency of body bends, reduced exploratory movements and reduced performance in an endurance exercise test. Notably, mitochondrial function was altered in the fahd-1(tm5005) mutant strain, as shown by a reduction of mitochondrial membrane potential and a reduced oxygen consumption of fahd-1(tm5005) animals. Mitochondrial dysfunction was accompanied by lifespan extension in worms grown at elevated temperature; however, unlike in mutant worms with a defect in the electron transport chain, the mitochondrial unfolded protein response was not upregulated in worms upon inactivation of fahd-1. Together these data establish a role of fahd-1 to maintain mitochondrial function and consequently physical activity in nematodes.