Measurement of membrane binding between recoverin,a calcium-myristoyl switch protein,and lipid bilayers by AFM-based force spectroscopy

Myristoyl switch is a feature of several peripheral membrane proteins involved in signal transduction pathways. This unique molecular property is best illustrated by the "Ca(2+)-myristoyl switch" of recoverin, which is a Ca(2+)-binding protein present in retinal rod cells of vertebrates. In this transduction pathway, the Ca(2+)-myristoyl switch acts as a calcium sensor involved in cell recovery from photoactivation. Ca(2+) binding by recoverin induces the extrusion of its myristoyl group to the solvent, which leads to its translocation from cytosol to rod disk membranes. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the extent of membrane binding of recoverin in the absence and presence of calcium, and to quantify this force of binding. An adhesion force of 48 +/- 5 pN was measured between recoverin and supported phospholipid bilayers in the presence of Ca(2+). However, no binding was observed in the absence of Ca(2+). Experiments with nonmyristoylated recoverin confirmed these observations. Our results are consistent with previously measured extraction forces of lipids from membranes.     

Identification of a novel saturable endoplasmic reticulum localization mechanism mediated by the C-terminus of a Dictyostelium protein disulfide isomerase

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.     

Flagellar switch of Salmonella typhimurium: gene sequences and deduced protein sequences.

The fliG, fliM, and fliN genes of Salmonella typhimurium encode flagellar components that participate in energy transduction and switching. We have cloned these genes and determined their sequences. The deduced amino acid sequences correspond to proteins with molecular masses of 36,809, 37,815, and 14,772 daltons, respectively. None of the protein sequences are especially hydrophobic or look as though they correspond to integral membrane proteins, a result consistent with other evidence suggesting that the proteins may be peripheral to the membrane, possibly mounted onto the basal body M ring. The fliL gene, which immediately precedes fliM, is of unknown function; it encodes a protein with a deduced molecular mass of 17,082 daltons. The hydropathy profile of FliL indicates that it is likely to be an integral membrane protein with at least one spanning segment, near its N terminus. None of the four proteins exhibit consensus N-terminal signal sequences. Comparison of the fliL, fliM, and fliN sequences with the homologous ones in Escherichia coli reveals ranges of similarities of 77 to 95% at the amino acid level and 75 to 86% at the nucleotide level, with the majority (58 to 89%) of codon changes being synonymous ones.     

Forespore engulfment mediated by a ratchet-like mechanism

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.     

Subcellular Golgi localization of stathmin family proteins is promoted by a specific set of DHHC palmitoyl transferases

Protein palmitoylation is a reversible lipid modification that plays critical roles in protein sorting and targeting to specific cellular compartments. The neuronal microtubule-regulatory phosphoproteins of the stathmin family (SCG10/stathmin 2, SCLIP/stathmin 3, and RB3/stathmin 4) are peripheral proteins that fulfill specific and complementary roles in the formation and maturation of the nervous system. All neuronal stathmins are localized at the Golgi complex and at vesicles along axons and dendrites. Their membrane anchoring results from palmitoylation of two close cysteine residues present within their homologous N-terminal targeting domains. By preventing palmitoylation with 2-bromopalmitate or disrupting the integrity of the Golgi with brefeldin A, we were able to show that palmitoylation of stathmins 2 and 3 likely occurs at the Golgi and is crucial for their specific subcellular localization and trafficking. In addition, this membrane binding is promoted by a specific set of palmitoyl transferases that localize with stathmins 2 and 3 at the Golgi, directly interact with them, and enhance their membrane association. The subcellular membrane-associated microtubule-regulatory activity of stathmins might then be fine-tuned by extracellular stimuli controlling their reversible palmitoylation, which can be viewed as a crucial regulatory process for specific and local functions of stathmins in neurons.     

Paraquat neurotoxicity is mediated by a Bak-dependent mechanism

Paraquat (PQ) causes selective degeneration of dopaminergic neurons in the substantia nigra pars compacta, reproducing an important pathological feature of Parkinson disease. Oxidative stress, c-Jun N-terminal kinase activation, and alpha-synuclein aggregation are each induced by PQ, but details of the cell death mechanisms involved remain unclear. We have identified a Bak-dependent cell death mechanism that is required for PQ-induced neurotoxicity. PQ induced morphological and biochemical features that were consistent with apoptosis, including dose-dependent cytochrome c release, with subsequent caspase-3 and poly(ADP-ribose) polymerase cleavage. Changes in nuclear morphology and loss of viability were blocked by cycloheximide, caspase inhibitor, and Bcl-2 overexpression. Evaluation of Bcl-2 family members showed that PQ induced high levels of Bak, Bid, BNip3, and Noxa. Small interfering RNA-mediated knockdown of BNip3, Noxa, and Bak each protected cells from PQ, but Bax knockdown did not. Finally, we tested the sensitivity of Bak-deficient mice and found them to be resistant to PQ treatments that depleted tyrosine hydroxylase immuno-positive neurons in the substantia nigra pars compacta of wild-type mice.     

Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation

Calcium-dependent protein kinases (CDPKs), the most abundant serine/threonine kinases in plants, are found in various subcellular localizations, which suggests that this family of kinases may be involved in multiple signal transduction pathways. A complete analysis to try to understand the molecular basis of the presence of CDPKs in various localizations in the cell has not been accomplished yet. It has been suggested that myristoylation may be responsible for membrane association of CDPKs. In this study, we used a rice CDPK, OSCPK2, which has a consensus sequence for myristoylation at the N-terminus, to address this question. We expressed wild-type OSCPK2 and various mutants in different heterologous systems to investigate the factors that affect its membrane association. The results show that OSCPK2 is myristoylated and palmitoylated and targeted to the membrane fraction. Both modifications are required, myristoylation being essential for membrane localization and palmitoylation for its full association. The fact that palmitoylation is a reversible modification may provide a mechanism for regulation of the subcellular localization. OSCPK2 is the first CDPK shown to be targeted to membranes by an src homology domain 4 (SH4) located at the N-terminus of the molecule.     

Nucleolar localization of GLTSCR2/PICT-1 is mediated by multiple unique nucleolar localization sequences

The human glioma tumor suppressor candidate region 2 gene product, GLTSCR2, also called 'protein interacting with carboxyl terminus 1' (PICT-1), has been implicated in the regulation of two major tumor suppressor proteins, PTEN and p53, and reported to bind the membrane-cytoskeleton regulator of cell signaling, Merlin. PICT-1 is a nucleolar protein, conserved among eukaryotes, and its yeast homolog has been functionally associated with ribosomal RNA processing. By means of confocal microscopy of EGFP and myc-tagged PICT-1 fusion proteins, we delineate that the nucleolar localization of PICT-1 is mediated by two independent nucleolar localization sequences (NoLS). Unlike most NoLSs, these NoLSs are relatively long with flexible boundaries and contain arginine and leucine clusters. In addition, we show that PICT-1 exhibits a nucleolar distribution similar to proteins involved in ribosomal RNA processing, yet does not colocalize precisely with either UBF1 or Fibrillarin under normal or stressed conditions. Identification of the precise location of PICT-1 and the signals that mediate its nucleolar localization is an important step towards advancing our understanding of the demonstrated influence of this protein on cell fate and tumorigenesis.     

G蛋白偶联受体激活丝裂原活化蛋白激酶的机理

多种Ｇ蛋白偶联受体的均能激活丝裂原活化蛋白激酶。Ｇｉ蛋白偶联受体主要通过其βγ亚基,依赖Ｒａｓ蛋白途径;在大多数哺乳类细胞中Ｇｓ蛋白偶联受体通过ＰＫＡ途径抑制Ｒａｓ依赖的ＭＡＰＫ活化,但在ＣＯＳ－７细胞,Ｇｓ蛋白偶联受体通过ＰＫＡ途径使表达的ＭＡＰＫ活化;Ｇｑ蛋白偶联受体主要通过ＰＫＣ途径依赖或非依赖于Ｒａｓ使ＭＡＰＫ活化。ＭＡＰＫ信号途径中ＥＧＦ受体,酪氨酸激酶及调节蛋白Ｓｈｃ等联级反应蛋白可能     

Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for Flagellar function

Reversion analysis of flagellar-motility mutants of Chlamydomonas reinhardtii yields an unusual class of intergenic suppressor mutations that restore flagellar activity to paralyzed radial-spoke or central-pair mutants without altering the structural or molecular defects associated with the original mutations. Four suppressors representing independent genetic loci were studied in detail. Two of the mutations, suppf1 and suppf2, restore flagellar motility to either radial-spoke or central-pair mutants of different genes. The mutants suppf3 and suppf 4 suppress flagellar paralysis associated only with mutants defective for the radial spokes. Analyses of the axonemal polypeptides of suppf1, suppf3 and suppf4 mutants indicate that the mutations restore flagellar activity to paralyzed radial-spoke or central-pair mutants by altering other components of the flagellar axoneme. suppf1 shows an altered electrophoretic migration for a 325,000 molecular weight polypeptide known to be a subunit of an outer-arm dynein. suppf3 and suppf4 are missing different axonemal polypeptides with molecular weights of 60,000 (in the case of suppf3), and 40,000 and 29,000 (in the case of suppf4). Genetic evidence has been obtained indicating that the polypeptides affected in suppf3 and suppf4 are components of a newly identified functional and/or structural compartment of the flagellar axoneme. The suppressor mutations described here reveal the operation of a control mechanism that inhibits the operations of flagellar movements in the presence of radial-spoke or central-pair defects. Suppressor mutations release the inhibition. The molecular defects of suppf1, suppf3 and suppf4 provide evidence that the inhibitory mechanism can be interrupted at two different levels of axonemal function.     

Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

Retinal progenitor cells （RPCs） are neural stem cells able to differentiate into any normal adult retinal cell type, except for pigment epithelial cells. Retinoic acid （RA） is a powerful growth/differentiation factor that generally causes growth inhibition, differentiation and/or apoptosis. In this study, we demonstrate that RA not only affects mouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affecting RPC proliferation. The enhanced cell survival was accompanied by a significant upregulation of the expression of protein kinase A （PKA） and several protein kinase C （PKC） isoforms. Treatment of cells grown in RA-free media with 8-bromoadenosine3＇,5＇-cyclic monophosphate, a known activator of PKA, resulted in an anti-apoptotic effect similar to that caused by RA; whereas the PKA inhibitor N-[2-（p-bromocinnamylamino）ethyl]-5-isoquinolinesul- fonamide dihydrochloride led to a significant （-32%） increase in apoptosis. In contrast, treatment of RPCs with any of two PKC selective inhibitors, 2,2＇,3,3＇,4,4＇-hexahydroxy-1,1 ＇-biphenyl-6,6＇-dimethanol dimethyl ether and bisindolylmaleimide XI, led to diminished apoptosis; while a PKC activator, phorbol 12-myristate 13-acetate, increased apoptosis. These and other data suggest that the effect of RA on RPC survival is mostly due to the increased anti-apoptotic activity elicited by PKA, which might in turn be antagonized by PKC. Such a mechanism is a new example of tight regulation of important biological processes triggered by RA. Although the detailed mechanisms remain to be elucidated, we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changed expression of p53 or bcl-2, and appears to be independent of 15-amyloid, Fas ligand, TNF-α, ganglioside GM1 and ceramide C 16-induced apoptotic pathways.     

Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism

The amount of dopamine transporter (DAT) present at the cell surface is rapidly regulated by the rates of DAT internalization to endosomes and DAT recycling back to the plasma membrane. The re-distribution of the transporter from the cell surface to endosomes was induced by phorbol ester activation of protein kinase C in porcine aortic endothelial cells stably expressing the human DAT. Inhibition of DAT recycling with the carboxylic ionophore monensin also caused significant accumulation of DAT in early endosomes and a concomitant loss of DAT from the cell surface, due to protein kinase C-independent constitutive internalization of DAT in the absence of recycling. Such monensin-induced relocation of DAT to endosomes was therefore utilized as a measure of the constitutive internalization of DAT. Knock-down of clathrin heavy chain or dynamin II by small interfering RNAs dramatically inhibited both constitutive and protein kinase C-mediated internalization of DAT. In contrast, neither monensin-dependent nor phorbol ester-induced re-distribution of DAT were affected by inhibitors of endocytosis through cholesterol-rich membrane microdomains. Mutational analysis revealed the potential importance of amino acid residues 587-597 in DAT internalization. Altogether, the data suggest that both constitutive and protein kinase C-mediated internalization of DAT utilize the clathrin-dependent endocytic pathway, but likely involve unconventional mechanisms.     

A mechanism for polar protein localization in bacteria

We investigate a mechanism for the polar localization of proteins in bacteria. We focus on the MinCD/DivIVA system regulating division site placement in the rod-shaped bacterium Bacillus subtilis. Our model relies on a combination of geometric effects and reaction-diffusion dynamics to direct proteins to both cell poles, where division is then blocked. We discuss similarities and differences with related division models in Escherichia coli and also develop extensions of the model to asymmetric polar protein localization. We propose that our mechanism for polar localization may be employed more widely in bacteria, especially in outgrowing spores, which do not possess any pre-existing polar division apparatus from prior division events.     

A novel mechanism for mitogen-activated protein kinase localization

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.     

Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells

Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.     

Modulation of protein kinase C activity by palmitoyl esters of maltose.

Mixtures of maltose palmitates containing predominantly maltose tetrapalmitate (designated MTP) possess immune potentiating and antitumor properties. Immune potentiation derives from macrophage activation and B lymphocyte mitogenicity and antitumor action from anti-angiogenic activity. Their mode of action at the cellular level is not known. Since high performance liquid chromatography (HPLC) provided purified maltose palmitates, we tested whether they individually and as a mixture could modulate activity of protein Kinase C (PKC), an enzyme implicated in mitogenic and release reactions. MTP activated crude lymphocyte and purified brain PKC in the absence of phosphatidyl serine (PS). It also augmented labeled dibutyryl phorbol (PDBu) binding to the brain enzyme in the absence of phospholipid. HPLC purified maltose tetrapalmitates (two isomers) were insoluble in aqueous solvent, and activated PKC slightly after incorporation into PS liposomes. Purified maltose di- and tri-palmitates were inhibitory to the enzyme. The activation of PKC was, therefore, due to higher saturated maltose palmitates, well dispersed by less substituted maltose palmitates acting as emulsifiers.     

Nucleolar localization of parafibromin is mediated by three nucleolar localization signals

Parafibromin is a putative tumor suppressor encoded by HRPT2 and implicated in parathyroid tumorigenesis. We previously reported a functional bipartite nuclear localization signal (NLS) at residues 125-139. We now demonstrate that parafibromin exhibits nucleolar localization, mediated by three nucleolar localization signals (NoLS) at resides 76-92, 192-194 and 393-409. These NoLS represent clusters of basic amino acids arginine and lysine, similar to those found in other nucleolar proteins, as well as being characteristic of NLSs. While parafibromin's bipartite NLS is the primary determinant of nuclear localization, it does not mediate nucleolar localization. In contrast, the three identified NoLSs play only a minor role in nuclear localization, but are critical for the nucleolar localization of parafibromin.     

Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses

RNA silencing is an evolutionarily conserved surveillance system that occurs in a broad range of eukaryotic organisms. In plants, RNA silencing acts as an antiviral system; thus, successful virus infection requires suppression of gene silencing. A number of viral suppressors have been identified so far; however, the molecular bases of silencing suppression are still poorly understood. Here we show that p19 of Cymbidium ringspot virus (CymRSV) inhibits RNA silencing via its small RNA-binding activity in vivo. Small RNAs bound by p19 in planta are bona fide double-stranded siRNAs and they are silencing competent in the in vitro RNA-silencing system. p19 also suppresses RNA silencing in the heterologous Drosophila in vitro system by preventing siRNA incorporation into RISC. During CymRSV infection, p19 markedly diminishes the amount of free siRNA in cells by forming p19-siRNA complexes, thus making siRNAs inaccessible for effector complexes of RNA-silencing machinery. Furthermore, the obtained results also suggest that the p19-mediated sequestration of siRNAs in virus-infected cells blocks the spread of the mobile, systemic signal of RNA silencing.