ATP-Dependent Steps in Peroxisomal Protein Import.

Peroxisomes are organelles that play a central role in lipid metabolism and cellular redox homeostasis. The import of peroxisomal matrix proteins by peroxisomal targeting signal (PTS) receptors is an ATP-dependent mechanism. However, the energy-dependent steps do not occur early during the binding of the receptor-cargo complex to the membrane but late, because they are linked to the peroxisomal export complex for the release of the unloaded receptor. The first ATP-demanding step is the cysteine-dependent monoubiquitination of the PTS receptors, which is required for recognition by the AAA+ peroxins. They execute the second ATP-dependent step by extracting the ubiqitinated PTS receptors from the membrane for release back to the cytosol. After deubiquitination, the PTS receptors regain import competence and can facilitate further rounds of cargo import. Here, we give a general overview and discuss recent data regarding the ATP-dependent steps in peroxisome protein import.

The twin-arginine protein translocation pathway.

The twin-arginine translocation (Tat) system, found in prokaryotes, chloroplasts, and some mitochondria, allows folded proteins to be moved across membranes. How this transport is achieved without significant ion leakage is an intriguing mechanistic question. Tat transport is mediated by complexes formed from small integral membrane proteins from just two protein families. Atomic-resolution structures have recently been determined for representatives of both these protein families, providing the first molecular-level glimpse of the Tat machinery. I review our current understanding of the mechanism of Tat transport in light of these new structural data.

Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.

A plant-unique protein BLISTER coordinates with core retromer to modulate endosomal sorting of plasma membrane and vacuolar proteins.

The retromer is a heteromeric protein complex that localizes to endosomal membranes and drives the formation of endosomal tubules that recycle membrane protein cargoes. In plants, the retromer plays essential and canonical functions in regulating the transport of vacuolar storage proteins and the recycle of endocytosed plasma membrane proteins (PM); however, the mechanisms underlying the regulation of assembly, protein stability, and membrane recruitment of the plant retromer complex remain to be elucidated. In this study, we identify a plant-unique endosomal regulator termed BLISTER (BLI), which colocalizes and associates with the retromer complex by interacting with the retromer core subunits VPS35 and VPS29. Depletion of BLI perturbs the assembly and membrane recruitment of the retromer core VPS26-VPS35-VPS29 trimer. Consequently, depletion of BLI disrupts retromer-regulated endosomal trafficking function, including transport of soluble vacuolar proteins and recycling of endocytosed PIN-FORMED (PIN) proteins from the endosomes back to the PM. Moreover, genetic analysis in Arabidopsis thaliana mutants reveals BLI and core retromer interact genetically in the regulation of endosomal trafficking. Taken together, we identified BLI as a plant-specific endosomal regulator, which functions in retromer pathway to modulate the recycling of endocytosed PM proteins and the trafficking of soluble vacuolar cargoes.

Molecular mechanism of GTP binding- and dimerization-induced enhancement of Sar1-mediated membrane remodeling.

The Sar1 GTPase initiates coat protein II (COPII)-mediated protein transport by generating membrane curvature at subdomains on the endoplasmic reticulum, where it is activated by the guanine nucleotide exchange factor (GEF) Sec12. Crystal structures of GDP- and GTP-bound forms of Sar1 suggest that it undergoes a conformational switch in which GTP binding enhances the exposure of an amino-terminal amphipathic helix necessary for efficient membrane penetration. However, key residues in the amino terminus were not resolved in crystal structures, and experimental studies have suggested that the amino terminus of Sar1 is solvent-exposed in the absence of a membrane, even in the GDP-bound state. Therefore, the molecular mechanism by which GTP binding activates the membrane-remodeling activity of Sar1 remains unclear. Using atomistic molecular dynamics simulations, we compare the membrane-binding and curvature generation activities of Sar1 in its GDP- and GTP-bound states. We show that in the GTP-bound state, Sar1 inserts into the membrane with its complete (residues 1 to 23) amphipathic amino-terminal helix, while Sar1-GDP binds to the membrane only through its first 12 residues. Such differential membrane-binding modes translate into significant differences in the protein volume inserted into the membrane. As a result, Sar1-GTP generates positive membrane curvature 10 to 20 times higher than Sar1-GDP. Dimerization of the GTP-bound form of Sar1 further amplifies curvature generation. Taken together, our results present a detailed molecular mechanism for how the nucleotide-bound state of Sar1 regulates its membrane-binding and remodeling activities in a concentration-dependent manner, paving the way toward a better understanding COPII-mediated membrane transport.

Cholangiocytes express an isoform of soluble adenylyl cyclase that is N-linked glycosylated and secreted in extracellular vesicles.

Soluble adenylyl cyclase (sAC)-derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85 ) or 75 kD (sAC75 ) sAC protein band under glucose-sufficient or glucose-deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase-F demonstrated that both sAC75 and sAC85 are N-linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo-H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N-linked glycosylated sAC (sACEV ) in extracellular vesicles under conditions that support intracellular sAC85 (glucose-sufficient) as opposed to sAC75 (glucose-deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N-linked glycosylated sAC isoform that is rapidly released into extracellular vesicles.

Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7.

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

BACKGROUND: Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. METHODS: Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). RESULTS: ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. CONCLUSIONS: ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Sengers Syndrome-Associated Mitochondrial Acylglycerol Kinase Is a Subunit of the Human TIM22 Protein Import Complex.

Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.

A unique bacterial secretion machinery with multiple secretion centers.

The Porphyromonas gingivalis type IX secretion system (T9SS) promotes periodontal disease by secreting gingipains and other virulence factors. By in situ cryoelectron tomography, we report that the P. gingivalis T9SS consists of 18 PorM dimers arranged as a large, caged ring in the periplasm. Near the outer membrane, PorM dimers interact with a PorKN ring complex of ∼52 nm in diameter. PorMKN translocation complexes of a given T9SS adopt distinct conformations energized by the proton motive force, suggestive of different activation states. At the inner membrane, PorM associates with a cytoplasmic complex that exhibits 12-fold symmetry and requires both PorM and PorL for assembly. Activated motors deliver substrates across the outer membrane via one of eight Sov translocons arranged in a ring. The T9SSs are unique among known secretion systems in bacteria and eukaryotes in their assembly as supramolecular machines composed of apparently independently functioning translocation motors and export pores.

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites.

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs-modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

CD14 recycling modulates LPS-induced inflammatory responses of murine macrophages.

TLR4 is activated by the bacterial endotoxin lipopolysaccharide (LPS) and triggers two proinflammatory signaling cascades: a MyD88-dependent one in the plasma membrane, and the following TRIF-dependent one in endosomes. An inadequate inflammatory reaction can be detrimental for the organism by leading to sepsis. Therefore, novel approaches to therapeutic modulation of TLR4 signaling are being sought after. The TLR4 activity is tightly connected with the presence of CD14, a GPI-anchored protein that transfers LPS monomers to the receptor and controls its endocytosis. In this study we focused on CD14 trafficking as a still poorly understood factor affecting TLR4 activity. Two independent assays were used to show that after endocytosis CD14 can recycle back to the plasma membrane in both unstimulated and stimulated cells. This route of CD14 trafficking can be controlled by sorting nexins (SNX) 1, 2 and 6, and is important for maintaining the surface level and the total level of CD14, but can also affect the amount of TLR4. Silencing of these SNXs attenuated especially the CD14-dependent endosomal signaling of TLR4, making them a new target for therapeutic regulation of the inflammatory response of macrophages to LPS.

Arabidopsis callus grafts: a system to study symplasmic intercellular macromolecular transport and gene silencing spread.

In the present study, we present callus grafting, comprising a method for reproducibly generating tissue chimeras from callus cultures of Arabidopsis thaliana. In this way, callus cultures of different genetic backgrounds may be co-cultivated such that cell-to-cell connectivity is achieved as a chimeric tissue is formed. To track intercellular connectivity and transport between non-clonal callus cells, we used transgenic lines expressing fluorescently tagged mobile and non-mobile fusion constructs. Using fluorescently-labelled reporter lines that label plasmodesmata, we show that secondary complex plasmodesmata are present at the cell walls of connected cells. We use this system to investigate cell-to-cell transport across the callus graft junction and show that different proteins and RNAs are mobile between non-clonal callus cells. Finally, we take advantage of the callus culture system to probe intercellular connectivity of grafted leaf and root calli and the effect of different light regimes of cell-to-cell transport. Taking advantage of the ability of callus to be cultivated in the complete absence of light, we show that the rate of silencing spread is significantly decreased in chimeric calli cultivated in total darkness. We propose that callus grafting is a fast and reliable method for analysing the capacity of a macromolecule to be exchanged between cells independent of the vasculature.

Yeast profilin mutants inhibit classical nuclear import and alter the balance between actin and tubulin levels.

Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.

Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.

Phase separation of components of ER exit sites (ERES) into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon exposure to specific cellular stressors, namely, salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Finally, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.

Construction of an orthogonal transport system for Saccharomyces cerevisiae peroxisome to efficiently produce sesquiterpenes.

Subcellular compartmentalization is a crucial evolution characteristic of eukaryotic cells, providing inherent advantages for the construction of artificial biological systems to efficiently produce natural products. The establishment of an artificial protein transport system represents a pivotal initial step towards developing efficient artificial biological systems. Peroxisome has been demonstrated as a suitable subcellular compartment for the biosynthesis of terpenes in yeast. In this study, an artificial protein transporter ScPEX5* was firstly constructed by fusing the N-terminal sequence of PEX5 from S. cerevisiae and the C-terminal sequence of PEX5. Subsequently, an artificial protein transport system including the artificial signaling peptide YQSYY and its enhancing upstream 9 amino acid (9AA) residues along with ScPEX5* was demonstrated to exhibit orthogonality to the internal transport system of peroxisomes in S. cerevisiae. Furthermore, a library of 9AA residues was constructed and selected using high throughput pigment screening system to obtain an optimized signaling peptide (oPTS1*). Finally, the ScPEX5*-oPTS1* system was employed to construct yeast cell factories capable of producing the sesquiterpene α-humulene, resulting in an impressive α-humulene titer of 17.33 g/L and a productivity of 0.22 g/L/h achieved through fed-batch fermentation in a 5 L bioreactor. This research presents a valuable tool for the construction of artificial peroxisome cell factories and effective strategies for synthesizing other natural products in yeast.

The role of chloroplast SRP54 domains and its C-terminal tail region in post- and cotranslational protein transport in vivo.

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear- as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

Effects of poly (ADP-ribose) polymerase 1 (PARP1) on silk proteins in the silkworm, Bombyx mori.

Animal silk is economically important, while silk secretion is a complex and subtle mechanism regulated by many genes. We identified the poly (ADP-ribose) polymerase (PARP1) gene of the silkworm and successfully cloned its coding sequence (CDS) sequence. Using clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) technology, we screened single guide RNA (sgRNA) with high knockout efficiency by cellular experiments and obtained PARP1 mutants by knocking out the PARP1 gene of the silkworm at the individual level. We found that the mutants mainly exhibited phenotypes such as smaller cocoon size and reduced cocoon shell rate than the wild type. We also detected the expression of silk protein genes in the mutant by quantitative real-time PCR (qPCR) and found that the expression of some silk protein genes was slightly down-regulated. Meanwhile, together with the results of transcriptomic analysis, we hypothesized that PARP1 may affect the synthesis of silk proteins, resulting in their failure to function properly. Our study may provide an important reference for future in-depth refinement of the molecular mechanism of silk protein expression in silk-producing animals, as well as a potential idea for future development of molecular breeding lines of silkworms to improve silk production.

Clinicopathological significance of unraveling mitochondrial pathway alterations in non-small-cell lung cancer.

Early detection, accurate monitoring, and therapeutics are major problems in non-small-cell lung cancer (NSCLC) patients. We identified genomic copy number variation of a unique panel of 40 mitochondria-targeted genes in NSCLCs (GEOGSE #29365). Validation of mRNA expression of these molecules revealed an altered panel of 34 genes in lung adenocarcinomas (LUAD) and 36 genes in lung squamous cell carcinomas (LUSC). In the LUAD subtype (n = 533), we identified 29 upregulated and 5 downregulated genes, while in the LUSC subtype (n = 502), a panel of 30 upregulated and 6 downregulated genes were discovered. The majority of these genes are associated with mitochondrial protein transport, ferroptosis, calcium signaling, metabolism, OXPHOS function, TCA cycle, apoptosis, and MARylation. Altered mRNA expression of SLC25A4, ACSF2, MACROD1, and GCAT was associated with poor survival of the NSCLC patients. Progressive loss of SLC25A4 protein expression was confirmed in NSCLC tissues (n = 59), predicting poor survival of the patients. Forced overexpression of SLC25A4 in two LUAD cell lines inhibited their growth, viability, and migration. A significant association of the altered mitochondrial pathway genes with LC subtype-specific classical molecular signatures was observed, implicating the existence of nuclear-mitochondrial cross-talks. Key alteration signatures shared between LUAD and LUSC subtypes including SLC25A4, ACSF2, MACROD1, MDH2, LONP1, MTHFD2, and CA5A could be helpful in developing new biomarkers and therapeutics.