Ceramide in Plasma Membrane Repair

The perforation of the plasmalemma by pore-forming toxins causes an influx of Ca2+ and an efflux of cytoplasmic proteins. In order to ensure cellular survival, lesions have to be identified, plugged and removed from the membrane. The Ca2+-driven fusion of lysosomes with the plasma membrane leads to hydrolysis of sphingomyelin by acid sphingomyelinase and a formation of ceramide platforms in the outer leaflet of the lipid bilayer. We propose that the negative curvature, promoted by tighter packing of lipids in the outer layer, leads to an inward vesiculation of the damaged area for its endocytotic uptake and internal degradation. In contrast, the activation of neutral sphingomyelinase triggers the production of ceramide within the inner leaflet of the lipid bilayer, thereby promoting an outward curvature, which enables the cell to shed the membrane-containing toxin pore into the extracellular space. In this process, ceramide is supported by members of the annexin protein family which act as Ca2+ sensors and as membrane fusion agents.

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial -barrel proteins specialized in mitochondrial genome inheritance

Mitochondria cannot form de novo but require mechanisms allowing their inheritance to daughter cells. In contrast to most other eukaryotes Trypanosoma brucei has a single mitochondrion whose single-unit genome is physically connected to the flagellum. Here we identify a β-barrel mitochondrial outer membrane protein, termed tripartite attachment complex 40 (TAC40), that localizes to this connection. TAC40 is essential for mitochondrial DNA inheritance and belongs to the mitochondrial porin protein family. However, it is not specifically related to any of the three subclasses of mitochondrial porins represented by the metabolite transporter voltage-dependent anion channel (VDAC), the protein translocator of the outer membrane 40 (TOM40), or the fungi-specific MDM10, a component of the endoplasmic reticulum–mitochondria encounter structure (ERMES). MDM10 and TAC40 mediate cellular architecture and participate in transmembrane complexes that are essential for mitochondrial DNA inheritance. In yeast MDM10, in the context of the ERMES, is postulated to connect the mitochondrial genomes to actin filaments, whereas in trypanosomes TAC40 mediates the linkage of the mitochondrial DNA to the basal body of the flagellum. However, TAC40 does not colocalize with trypanosomal orthologs of ERMES components and, unlike MDM10, it regulates neither mitochondrial morphology nor the assembly of the protein translocase. TAC40 therefore defines a novel subclass of mitochondrial porins that is distinct from VDAC, TOM40, and MDM10. However, whereas the architecture of the TAC40-containing complex in trypanosomes and the MDM10-containing ERMES in yeast is very different, both are organized around a β-barrel protein of the mitochondrial porin family that mediates a DNA–cytoskeleton linkage that is essential for mitochondrial DNA inheritance.

AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis

For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids. A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant. AtPTR1 mediated growth of S. cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin. The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions. AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond. However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1. At pH 5.5 AtPTR1 had an apparent lower affinity (K-0.5 = 416 mum) for Ala-Asp compared with Ala-Ala (K-0.5 = 54 mum) and Ala-Lys (K-0.5 = 112 mum). Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane. In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides.

Effect of combined drought and heat stress on heat shock proteins in wheat varieties

Introduction: The re sponse of crop plants ex posed on drought or heat shock is related to de crease in the synthesis of normal proteins, accompanied by increased translation of heat shock proteins (HSPs). Though drought and heat stress have been studied individually, little is known about their combined effect on plants. Methods: The wheat (Triticum aestivum L.) varieties (Katya-tolerant, Sadovo or Mladka-susceptible) were potted in soil. Eight-day-old plants were ex posed to with drawing water for seven days. Heat shock was realized in growth chamber at 40 °C for 6h. A combination of drought and heat shock was per formed by subjecting drought-stressed plants to heat shock treatment. Expression of HSPs in the first leaf of wheat varieties was analyzed by SDS electrophoresis and immunoblotting. Polyclonal antibodies against HSP20, HSP60, HSP110 and mononclonal antibodies against HSP70 were used to distinguish the mentioned HSPs. Results: The leaf relative water content (RWC), which indicated the level of plant dehydration decreased significantly (34 %) under drought stressed conditions The electrolyte leakage of ions (EL), representing the level of the cell membrane stability in creased mark edly (68 %), especially under combination of drought and heat. Maximum EL was ob served in drought susceptible varieties Sadovo and Mladka. Drought and heat shock combination in the wheat plants resulted in the induction of specific HSPs. Conclusions: Our results demonstrate that the response of the wheat plants to a combination of drought and heat stress is different from the response of plants to each of these stresses applied separately. Induction of synergetic effect on HSP expression in case of combination between drought and heat was discussed in the case of two contrasting wheat varieties.

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.

Cloning and characterization of an Actinobacillus pleuropneumoniae outer membrane protein belonging to the family of PAL lipoproteins

A 14-kDa outer membrane protein (OMP) was purified from Actinobacillus pleuro-pneumoniae serotype 2. The protein strongly reacts with sera from pigs experimentally or naturally infected with any of the 12 serotypes of A. pleuropneumoniae. The gene encoding this protein was isolated from a gene library of A. pleuropneumoniae serotype 2 reference strain by immunoscreening. Expression of the cloned gene in Escherichia coli revealed that the protein is also located in the outer membrane fraction of the recombinant host. DNA sequence analysis of the gene reveals high similarity of the protein's amino acid sequence to that of the E. coli peptidoglycan-associated lipoprotein PAL, to the Haemophilus influenzae OMP P6 and to related proteins of several other Gram-negative bacteria. We have therefore named the 14-kDa protein PalA, and its corresponding gene, palA. The 20 amino-terminal amino acid residues of PalA constitute a signal sequence characteristic of membrane lipoproteins of prokaryotes with a recognition site for the signal sequence peptidase II and a sorting signal for the final localization of the mature protein in the outer membrane. The DNA sequence upstream of palA contains an open reading frame which is highly similar to the E. coli tolB gene, indicating a gene cluster in A. pleuropneumoniae which is very similar to the E. coli tol locus. The palA gene is conserved and expressed in all A. pleuropneumoniae serotypes and in A. lignieresii. A very similar palA gene is present in A. suis and A. equuli.

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.

Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

A subclass of eukaryotic proteins is subject to modification with fatty acids, the most common of which are palmitic and myristic acid. Protein acylation allows association with cellular membranes in the absence of transmembrane domains. Here we examine POMP39, a protein previously described to be present in the outer mitochondrial membrane proteome (POMP) of the protozoan parasite Trypanosoma brucei. POMP39 lacks canonical transmembrane domains, but is likely both myristoylated and palmitoylated on its N-terminus. Interestingly, the protein is also dually localized on the surface of the mitochondrion as well as in the flagellum of both insect-stage and the bloodstream form of the parasites. Upon abolishing of global protein acylation or mutation of the myristoylation site, POMP39 relocates to the cytosol. RNAi-mediated ablation of the protein neither causes a growth phenotype in insect-stage nor bloodstream form trypanosomes.

TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Their protein import systems are of similar complexity yet different composition than those in other eukaryotes. To elucidate the composition of the trypanosomal translocase of the inner mitochondrial membrane (TIM) we performed CoIPs of epitope-tagged TbTim17 and two other candidates in combination with SILAC-based quantitative mass spectrometry. This led to the identification of ten candidates for core TIM subunits. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a trypanosomatid-specific 42 kDa protein (Tim42) and two putative orthologues of inactive rhomboid proteases were analyzed further. All three proteins are essential in both life cycle stages and their ablation results in a strong protein import defect in vivo and in vitro. Blue native PAGE revealed their presence in a high molecular weight complex. Unlike anticipated, trypanosomes have a highly complex TIM translocase that has extensively been redesigned. None of the three novel TIM subunits has ever been associated with mitochondrial protein import. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. This suggests an exciting analogy between protein translocases of mitochondria and the ER.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 μM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Development of a Novel Green Fluorescent Protein-Based Binding Assay to Study the Association of Plakins with Intermediate Filament Proteins.

Protein-protein interactions are fundamental for most biological processes, such as the formation of cellular structures and enzymatic complexes or in signaling pathways. The identification and characterization of protein-protein interactions are therefore essential for understanding the mechanisms and regulation of biological systems. The organization and dynamics of the cytoskeleton, as well as its anchorage to specific sites in the plasma membrane and organelles, are regulated by the plakins. These structurally related proteins anchor different cytoskeletal networks to each other and/or to other cellular structures. The association of several plakins with intermediate filaments (IFs) is critical for maintenance of the cytoarchitecture. Pathogenic mutations in the genes encoding different plakins can lead to dramatic manifestations, occurring principally in the skin, striated muscle, and/or nervous system, due to cytoskeletal disorganization resulting in abnormal cell fragility. Nevertheless, it is still unclear how plakins bind to IFs, although some general rules are slowly emerging. We here describe in detail a recently developed protein-protein fluorescence binding assay, based on the production of recombinant proteins tagged with green fluorescent protein (GFP) and their use as fluid-phase fluorescent ligands on immobilized IF proteins. Using this method, we have been able to assess the ability of C-terminal regions of GFP-tagged plakin proteins to bind to distinct IF proteins and IF domains. This simple and sensitive technique, which is expected to facilitate further studies in this area, can also be potentially employed for any kind of protein-protein interaction studies.