Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.     

Interaction of Nascent Chains with the Ribosomal Tunnel Proteins Rpl4, Rpl17, and Rpl39 of Saccharomyces cerevisiae

As translation proceeds, nascent polypeptides pass through an exit tunnel that traverses the large ribosomal subunit. Three ribosomal proteins, termed Rpl4, Rpl17, and Rpl39 expose domains to the interior of the exit tunnel of eukaryotic ribosomes. Here we generated ribosome-bound nascent chains in a homologous yeast translation system to analyze contacts between the tunnel proteins and nascent chains. As model proteins we employed Dap2, which contains a hydrophobic signal anchor (SA) segment, and the chimera Dap2α, in which the SA was replaced with a hydrophilic segment, with the propensity to form an α-helix. Employing a newly developed FLAG exposure assay, we find that the nascent SA segment but not the hydrophilic segment adopted a stable, α-helical structure within the tunnel when the most C-terminal SA residue was separated by 14 residues from the peptidyl transferase center. Using UV cross-linking, antibodies specifically recognizing Rpl17 or Rpl39, and a His₆-tagged version of Rpl4, we established that all three tunnel proteins of yeast contact the SA, whereas only Rpl4 and Rpl39 also contact the hydrophilic segment. Consistent with the localization of the tunnel exposed domains of Rpl17 and Rpl39, the SA was in contact with Rpl17 in the middle region and with Rpl39 in the exit region of the tunnel. In contrast, Rpl4 was in contact with nascent chain residues throughout the ribosomal tunnel.     

A Subunit of Eukaryotic Translation Initiation Factor 2α-Phosphatase (CreP/PPP1R15B) Regulates Membrane Traffic

The constitutive reverter of eIF2α phosphorylation (CReP)/PPP1r15B targets the catalytic subunit of protein phosphatase 1 (PP1c) to phosphorylated eIF2α (p-eIF2α) to promote its dephosphorylation and translation initiation. Here, we report a novel role and mode of action of CReP. We found that CReP regulates uptake of the pore-forming Staphylococcus aureus α-toxin by epithelial cells. This function was independent of PP1c and translation, although p-eIF2α was involved. The latter accumulated at sites of toxin attack and appeared conjointly with α-toxin in early endosomes. CReP localized to membranes, interacted with phosphomimetic eIF2α, and, upon overexpression, induced and decorated a population of intracellular vesicles, characterized by accumulation of N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE), a lipid marker of exosomes and intralumenal vesicles of multivesicular bodies. By truncation analysis, we delineated the CReP vesicle induction/association region, which comprises an amphipathic α-helix and is distinct from the PP1c interaction domain. CReP was also required for exocytosis from erythroleukemia cells and thus appears to play a broader role in membrane traffic. In summary, the mammalian traffic machinery co-opts p-eIF2α and CReP, regulators of translation initiation.     

αA-Crystallin and αB-Crystallin Reside in Separate Subcellular Compartments in the Developing Ocular Lens

αA-Crystallin (αA) and αB-crystallin (αB), the two prominent members of the small heat shock family of proteins are considered to be two subunits of one multimeric protein, α-crystallin, within the ocular lens. Outside of the ocular lens, however, αA and αB are known to be two independent proteins, with mutually exclusive expression in many tissues. This dichotomous view is buoyed by the high expression of αA and αB in the lens and their co-fractionation from lens extracts as one multimeric entity, α-crystallin. To understand the biological function(s) of each of these two proteins, it is important to investigate the biological basis of this perceived dichotomy; in this report, we address the question whether αA and αB exist as independent proteins in the ocular lens. Discontinuous sucrose density gradient fractionation and immunoconfocal localization reveal that in early developing rat lens αA is a membrane-associated small heat shock protein similar to αB but with remarkable differences. Employing an established protocol, we demonstrate that αB predominantly sediments with rough endoplasmic reticulum, whereas αA fractionates with smooth membranes. These biochemical observations were corroborated with immunogold labeling and transmission electron microscopy. Importantly, in the rat heart also, which does not contain αA, αB fractionates with rough endoplasmic reticulum, suggesting that αA has no influence on the distribution of αB. These data demonstrate presence of αA and αB in two separate subcellular membrane compartments, pointing to their independent existence in the developing ocular lens.     

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

All peripheral membrane proteins must negotiate unique constraints intrinsic to the biological interface of lipid bilayers and the cytosol. Phospholipase C-β (PLC-β) isozymes hydrolyze the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to propagate diverse intracellular responses that underlie the physiological action of many hormones, neurotransmitters, and growth factors. PLC-β isozymes are autoinhibited, and several proteins, including Gα_q, Gβγ, and Rac1, directly engage distinct regions of these phospholipases to release autoinhibition. To understand this process, we used a novel, soluble analog of PIP₂ that increases in fluorescence upon cleavage to monitor phospholipase activity in real time in the absence of membranes or detergents. High concentrations of Gα_q or Gβ₁γ₂ did not activate purified PLC-β3 under these conditions despite their robust capacity to activate PLC-β3 at membranes. In addition, mutants of PLC-β3 with crippled autoinhibition dramatically accelerated the hydrolysis of PIP₂ in membranes without an equivalent acceleration in the hydrolysis of the soluble analog. Our results illustrate that membranes are integral for the activation of PLC-β isozymes by diverse modulators, and we propose a model describing membrane-mediated allosterism within PLC-β isozymes.     

Feasibility of β-Sheet Breaker Peptide-H102 Treatment for Alzheimer's Disease Based on β-Amyloid Hypothesis

β-amyloid hypothesis is the predominant hypothesis in the study of pathogenesis of Alzheimer''s disease. This hypothesis claims that aggregation and neurotoxic effects of amyloid β (Aβ) is the common pathway in a variety of etiological factors for Alzheimer''s disease. Aβ peptide derives from amyloid precursor protein (APP). β-sheet breaker peptides can directly prevent and reverse protein misfolding and aggregation in conformational disorders. Based on the stereochemical structure of Aβ1-42 and aggregation character, we had designed a series of β-sheet breaker peptides in our previous work and screened out a 10-residue peptide β-sheet breaker peptide, H102. We evaluated the effects of H102 on expression of P-tau, several associated proteins, inflammatory factors and apoptosis factors, and examined the cognitive ability of APP transgenic mice by behavioral test. This study aims to validate the β-amyloid hypothesis and provide an experimental evidence for the feasibility of H102 treatment for Alzheimer''s disease.     

Annular Anionic Lipids Stabilize the Integrin αIIbβ3 Transmembrane Complex

Cationic membrane-proximal amino acids determine the topology of membrane proteins by interacting with anionic lipids that are restricted to the intracellular membrane leaflet. This mechanism implies that anionic lipids interfere with electrostatic interactions of membrane proteins. The integrin αIIbβ3 transmembrane (TM) complex is stabilized by a membrane-proximal αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) interaction; here, we examine the influence of anionic lipids on this complex. Anionic lipids compete for αIIb(Arg⁹⁹⁵) contacts with β3(Asp⁷²³) but paradoxically do not diminish the contribution of αIIb(Arg⁹⁹⁵)-β3(Asp⁷²³) to TM complex stability. Overall, anionic lipids in annular positions stabilize the αIIbβ3 TM complex by up to 0.50 ± 0.02 kcal/mol relative to zwitterionic lipids in a headgroup structure-dependent manner. Comparatively, integrin receptor activation requires TM complex destabilization of 1.5 ± 0.2 kcal/mol, revealing a sizeable influence of lipid composition on TM complex stability. We implicate changes in lipid headgroup accessibility to small molecules (physical membrane characteristics) and specific but dynamic protein-lipid contacts in this TM helix-helix stabilization. Thus, anionic lipids in ubiquitous annular positions can benefit the stability of membrane proteins while leaving membrane-proximal electrostatic interactions intact.     

Role of LARP6 and Nonmuscle Myosin in Partitioning of Collagen mRNAs to the ER Membrane

Type I collagen is extracellular matrix protein composed of two α1(I) and one α2(I) polypeptides that fold into triple helix. Collagen polypeptides are translated in coordination to synchronize the rate of triple helix folding to the rate of posttranslational modifications of individual polypeptides. This is especially important in conditions of high collagen production, like fibrosis. It has been assumed that collagen mRNAs are targeted to the membrane of the endoplasmic reticulum (ER) after translation of the signal peptide and by signal peptide recognition particle (SRP). Here we show that collagen mRNAs associate with the ER membrane even when translation is inhibited. Knock down of LARP6, an RNA binding protein which binds 5′ stem-loop of collagen mRNAs, releases a small amount of collagen mRNAs from the membrane. Depolimerization of nonmuscle myosin filaments has a similar, but stronger effect. In the absence of LARP6 or nonmuscle myosin filaments collagen polypeptides become hypermodified, are poorly secreted and accumulate in the cytosol. This indicates lack of coordination of their synthesis and retro-translocation due to hypermodifications and misfolding. Depolimerization of nonmuscle myosin does not alter the secretory pathway through ER and Golgi, suggesting that the role of nonmuscle myosin is primarily to partition collagen mRNAs to the ER membrane. We postulate that collagen mRNAs directly partition to the ER membrane prior to synthesis of the signal peptide and that LARP6 and nonmuscle myosin filaments mediate this process. This allows coordinated initiation of translation on the membrane bound collagen α1(I) and α2(I) mRNAs, a necessary step for proper synthesis of type I collagen.     

Rotavirus infection induces the phosphorylation of eIF2alpha but prevents the formation of stress granules

Early during the infection process, rotavirus causes the shutoff of cell protein synthesis, with the nonstructural viral protein NSP3 playing a vital role in the phenomenon. In this work, we have found that the translation initiation factor 2α (eIF2α) in infected cells becomes phosphorylated early after virus infection and remains in this state throughout the virus replication cycle, leading to a further inhibition of cell protein synthesis. Under these restrictive conditions, however, the viral proteins and some cellular proteins are efficiently translated. The phosphorylation of eIF2α was shown to depend on the synthesis of three viral proteins, VP2, NSP2, and NSP5, since in cells in which the expression of any of these three proteins was knocked down by RNA interference, the translation factor was not phosphorylated. The modification of this factor is, however, not needed for the replication of the virus, since mutant cells that produce a nonphosphorylatable eIF2α sustained virus replication as efficiently as wild-type cells. In uninfected cells, the phosphorylation of eIF2α induces the formation of stress granules, aggregates of stalled translation complexes that prevent the translation of mRNAs. In rotavirus-infected cells, even though eIF2α is phosphorylated these granules are not formed, suggesting that the virus prevents the assembly of these structures to allow the translation of its mRNAs. Under these conditions, some of the cellular proteins that form part of these structures were found to change their intracellular localization, with some of them having dramatic changes, like the poly(A) binding protein, which relocates from the cytoplasm to the nucleus in infected cells, a relocation that depends on the viral protein NSP3.     

COPS5 (Jab1) Protein Increases β Site Processing of Amyloid Precursor Protein and Amyloid β Peptide Generation by Stabilizing RanBP9 Protein Levels

Increased processing of amyloid precursor protein (APP) and accumulation of neurotoxic amyloid β peptide (Aβ) in the brain is central to the pathogenesis of Alzheimer''s disease (AD). Therefore, the identification of molecules that regulate Aβ generation is crucial for future therapeutic approaches for AD. We demonstrated previously that RanBP9 regulates Aβ generation in a number of cell lines and primary neuronal cultures by forming tripartite protein complexes with APP, low-density lipoprotein-related protein, and BACE1, consequently leading to increased amyloid plaque burden in the brain. RanBP9 is a scaffold protein that exists and functions in multiprotein complexes. To identify other proteins that may bind RanBP9 and regulate Aβ levels, we used a two-hybrid analysis against a human brain cDNA library and identified COPS5 as a novel RanBP9-interacting protein. This interaction was confirmed by coimmunoprecipitation experiments in both neuronal and non-neuronal cells and mouse brain. Colocalization of COPS5 and RanBP9 in the same subcellular compartments further supported the interaction of both proteins. Furthermore, like RanBP9, COPS5 robustly increased Aβ generation, followed by increased soluble APP-β (sAPP-β) and decreased soluble-APP-α (sAPP-α) levels. Most importantly, down-regulation of COPS5 by siRNAs reduced Aβ generation, implying that endogenous COPS5 regulates Aβ generation. Finally, COPS5 levels were increased significantly in AD brains and APΔE9 transgenic mice, and overexpression of COPS5 strongly increased RanBP9 protein levels by increasing its half-life. Taken together, these results suggest that COPS5 increases Aβ generation by increasing RanBP9 levels. Thus, COPS5 is a novel RanBP9-binding protein that increases APP processing and Aβ generation by stabilizing RanBP9 protein levels.     

Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics

β-site APP cleaving enzyme 1 (BACE1) is a transmembrane aspartyl protease with a lumenal active site that sheds the ectodomains of membrane proteins through juxtamembrane proteolysis. BACE1 has been studied principally for its role in Alzheimer''s disease as the β-secretase responsible for generating the amyloid-β protein. Emerging evidence from mouse models has identified the importance of BACE1 in myelination and cognitive performance. However, the substrates that BACE1 processes to regulate these functions are unknown, and to date only a few β-secretase substrates have been identified through candidate-based studies. Using an unbiased approach to substrate identification, we performed quantitative proteomic analysis of two human epithelial cell lines stably expressing BACE1 and identified 68 putative β-secretase substrates, a number of which we validated in a cell culture system. The vast majority were of type I transmembrane topology, although one was type II and three were GPI-linked proteins. Intriguingly, a preponderance of these proteins are involved in contact-dependent intercellular communication or serve as receptors and have recognized roles in the nervous system and other organs. No consistent sequence motif predicting BACE1 cleavage was identified in substrates versus non-substrates. These findings expand our understanding of the proteins and cellular processes that BACE1 may regulate, and suggest possible mechanisms of toxicity arising from chronic BACE1 inhibition.     

Ca2+-binding Motif of βγ-Crystallins

βγ-Crystallin-type double clamp (N/D)(N/D)XX(S/T)S motif is an established but sparsely investigated motif for Ca²⁺ binding. A βγ-crystallin domain is formed of two Greek key motifs, accommodating two Ca²⁺-binding sites. βγ-Crystallins make a separate class of Ca²⁺-binding proteins (CaBP), apparently a major group of CaBP in bacteria. Paralleling the diversity in βγ-crystallin domains, these motifs also show great diversity, both in structure and in function. Although the expression of some of them has been associated with stress, virulence, and adhesion, the functional implications of Ca²⁺ binding to βγ-crystallins in mediating biological processes are yet to be elucidated.     

Crystal Structures and RNA-binding Properties of the RNA Recognition Motifs of Heterogeneous Nuclear Ribonucleoprotein L: INSIGHTS INTO ITS ROLES IN ALTERNATIVE SPLICING REGULATION

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is an abundant RNA-binding protein implicated in many bioprocesses, including pre-mRNA processing, mRNA export of intronless genes, internal ribosomal entry site-mediated translation, and chromatin modification. It contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In this study, surface plasmon resonance spectroscopy assays revealed that all four RRM domains contribute to RNA binding. Furthermore, we elucidated the crystal structures of hnRNP L RRM1 and RRM34 at 2.0 and 1.8 Å, respectively. These RRMs all adopt the typical β1α1β2β3α2β4 topology, except for an unusual fifth β-strand in RRM3. RRM3 and RRM4 interact intimately with each other mainly through helical surfaces, leading the two β-sheets to face opposite directions. Structure-based mutations and surface plasmon resonance assay results suggested that the β-sheets of RRM1 and RRM34 are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays, together with cross-linking and dynamic light scattering assays, demonstrated that hnRNP L RRM34 facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and isothermal titration calorimetry binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative splicing regulation as an activator or repressor.     

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane

β₂-Adrenergic receptors (β₂-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β₂-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β₂-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β₂-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β₂-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β₂-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.     

Rab3-interacting Molecule γ Isoforms Lacking the Rab3-binding Domain Induce Long Lasting Currents but Block Neurotransmitter Vesicle Anchoring in Voltage-dependent P/Q-type Ca2+ Channels

Assembly of voltage-dependent Ca²⁺ channels (VDCCs) with their associated proteins regulates the coupling of VDCCs with upstream and downstream cellular events. Among the four isoforms of the Rab3-interacting molecule (RIM1 to -4), we have previously reported that VDCC β-subunits physically interact with the long α isoform of the presynaptic active zone scaffolding protein RIM1 (RIM1α) via its C terminus containing the C₂B domain. This interaction cooperates with RIM1α-Rab3 interaction to support neurotransmitter exocytosis by anchoring vesicles in the vicinity of VDCCs and by maintaining depolarization-triggered Ca²⁺ influx as a result of marked inhibition of voltage-dependent inactivation of VDCCs. However, physiological functions have not yet been elucidated for RIM3 and RIM4, which exist only as short γ isoforms (γ-RIMs), carrying the C-terminal C₂B domain common to RIMs but not the Rab3-binding region and other structural motifs present in the α-RIMs, including RIM1α. Here, we demonstrate that γ-RIMs also exert prominent suppression of VDCC inactivation via direct binding to β-subunits. In the pheochromocytoma PC12 cells, this common functional feature allows native RIMs to enhance acetylcholine secretion, whereas γ-RIMs are uniquely different from α-RIMs in blocking localization of neurotransmitter-containing vesicles near the plasma membrane. γ-RIMs as well as α-RIMs show wide distribution in central neurons, but knockdown of γ-RIMs attenuated glutamate release to a lesser extent than that of α-RIMs in cultured cerebellar neurons. The results suggest that sustained Ca²⁺ influx through suppression of VDCC inactivation by RIMs is a ubiquitous property of neurons, whereas the extent of vesicle anchoring to VDCCs at the plasma membrane may depend on the competition of α-RIMs with γ-RIMs for VDCC β-subunits.     

Isoform-selective Oligomer Formation of Saccharomyces cerevisiae p24 Family Proteins

p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER.     

Structural Insights of tBid,the Caspase-8-activated Bid,and Its BH3 Domain

The Bcl-2 family proteins regulate mitochondria-mediated apoptosis through intricate molecular mechanisms. One of the pro-apoptotic proteins, tBid, can induce apoptosis by promoting Bax activation, Bax homo-oligomerization, and mitochondrial outer membrane permeabilization. Association of tBid on the mitochondrial outer membrane is key to its biological function. Therefore knowing the conformation of tBid on the membrane will be the first step toward understanding its crucial role in triggering apoptosis. Here, we present NMR characterization of the structure and dynamics of human tBid in 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] micelles. Our data showed that tBid is monomeric with six well defined α-helices in the micelles. Compared with the full-length Bid structure, a longer flexible loop between tBid helix α₄ and α₅ was observed. Helices in tBid do not pack into a compact-fold but form an extended structure with a C-shape configuration in the micelles. All six tBid helices were shown to interact with LPPG micelles, with helix α₆ and α₇ being more embedded. Of note, the BH₃-containing helix α₃, which was previously believed to be exposed above the membrane surface, is also membrane associated, suggesting an “on the membrane” binding mode for tBid interaction with Bax. Our data provided structural details on the membrane-associated state of tBid and the functional implications of its membrane-associated BH₃ domain.