The C Terminus of the Alb3 Membrane Insertase Recruits cpSRP43 to the Thylakoid Membrane

The YidC/Oxa1/Alb3 family of membrane proteins controls the insertion and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here we describe the molecular mechanisms underlying the interaction of Alb3 with the chloroplast signal recognition particle (cpSRP). The Alb3 C-terminal domain (A3CT) is intrinsically disordered and recruits cpSRP to the thylakoid membrane by a coupled binding and folding mechanism. Two conserved, positively charged motifs reminiscent of chromodomain interaction motifs in histone tails are identified in A3CT that are essential for the Alb3-cpSRP43 interaction. They are absent in the C-terminal domain of Alb4, which therefore does not interact with cpSRP43. Chromodomain 2 in cpSRP43 appears as a central binding platform that can interact simultaneously with A3CT and cpSRP54. The observed negative cooperativity of the two binding events provides the first insights into cargo release at the thylakoid membrane. Taken together, our data show how Alb3 participates in cpSRP-dependent membrane targeting, and our data provide a molecular explanation why Alb4 cannot compensate for the loss of Alb3. Oxa1 and YidC utilize their positively charged, C-terminal domains for ribosome interaction in co-translational targeting. Alb3 is adapted for the chloroplast-specific Alb3-cpSRP43 interaction in post-translational targeting by extending the spectrum of chromodomain interactions.     

ROOT HAIR DEFECTIVE3 Family of Dynamin-Like GTPases Mediates Homotypic Endoplasmic Reticulum Fusion and Is Essential for Arabidopsis Development

In all eukaryotic cells, the endoplasmic reticulum (ER) forms a tubular network whose generation requires the fusion of ER membranes. In Arabidopsis (Arabidopsis thaliana), the membrane-bound GTPase ROOT HAIR DEFECTIVE3 (RHD3) is a potential candidate to mediate ER fusion. In addition, Arabidopsis has two tissue-specific isoforms of RHD3, namely RHD3-like (RL) proteins, and their function is not clear. Here, we show that a null allele of RHD3, rhd3-8, causes growth defects and shortened root hairs. A point mutant, rhd3-1, exhibits a more severe growth phenotype than the null mutant, likely because it exerts a dominant-negative effect on the RL proteins. Genetic analysis reveals that the double deletion of RHD3 and RL1 is lethal and that the rhd3 rl2 plants produce no viable pollen, suggesting that the RL proteins are redundant to RHD3. RHD3 family proteins can replace Sey1p, the homolog of RHD3 in yeast (Saccharomyces cerevisiae), in the maintenance of ER morphology, and they are able to fuse membranes both in vivo and in vitro. Our results suggest that RHD3 proteins mediate ER fusion and are essential for plant development and that the formation of the tubular ER network is of general physiological significance.In all eukaryotic cells, the endoplasmic reticulum (ER) comprises a continuous membrane system of sheets and tubules (Baumann and Walz, 2001; Shibata et al., 2006). ER tubules frequently connect through homotypic membrane fusion to form a reticular network (Lee and Chen, 1988; Prinz et al., 2000; Du et al., 2004). ER fusion in metazoans is mediated by the atlastins (ATLs), a class of dynamin-like, membrane-bound GTPases (Hu et al., 2009; Orso et al., 2009). ATL possesses a cytoplasmic N-terminal GTPase domain, followed by a helical domain, two closely spaced transmembrane domains, and a C-terminal cytosolic tail. ATL proteins localize mostly to ER tubules and they interact with the tubule-shaping proteins, reticulons and DP1 (Hu et al., 2009). A role for the ATLs in ER fusion is suggested by the fact that depletion of ATLs leads to long, nonbranched ER tubules in cultured cells (Hu et al., 2009) and to ER fragmentation in Drosophila melanogaster (Orso et al., 2009), possibly due to insufficient fusion between the tubules. Nonbranched ER tubules are also observed upon the expression of dominant-negative ATL mutants (Hu et al., 2009). In addition, antibodies to ATL inhibit ER network formation in Xenopus laevis egg extracts (Hu et al., 2009). Moreover, proteoliposomes containing purified D. melanogaster ATL undergo GTP-dependent fusion in vitro (Orso et al., 2009; Bian et al., 2011). The physiological significance of ER fusion is supported by the observation that mutations in human ATL1, the dominant isoform in the brain, cause hereditary spastic paraplegia (Zhao et al., 2001), a neurodegenerative disease characterized by axon shortening in corticospinal motor neurons and progressive spasticity and weakness of the lower limbs (Salinas et al., 2008).Many organisms lack ATL homologs. In yeast (Saccharomyces cerevisiae), another dynamin-like GTPase, Sey1p, has been found to share the same signature motifs and membrane topology as ATL (Hu et al., 2009). Recent work suggests that Sey1p mediates ER membrane fusion both in vivo and in vitro (Anwar et al., 2012). Cells lacking Sey1p grow normally (Hu et al., 2009), but additional mutation of an ER SNARE Ufe1p, which probably represents an alternative ER fusion mechanism in yeast, causes severe growth defects (Anwar et al., 2012). In Arabidopsis (Arabidopsis thaliana), the potential functional ortholog of ATL appears to be ROOT HAIR DEFECTIVE3 (RHD3; Hu et al., 2009), which was initially discovered by a genetic screen of root hair-defective mutants (Schiefelbein and Somerville, 1990). It is sequence related to Sey1p over the entire length (Wang et al., 1997; Brands and Ho, 2002). Mutations of RHD3 cause short and wavy root hairs (Schiefelbein and Somerville, 1990; Wang et al., 1997; Stefano et al., 2012) and defects in cell expansion (Wang et al., 2002).Despite the sequence homology between Sey1p and RHD3, it was reported that Sey1p could not replace RHD3 in plants and vice versa (Chen et al., 2011). Therefore, it is not clear whether RHD3 can mediate ER fusion. Another complication in plants is that the Arabidopsis RHD3 family also contains two RHD3-like (RL) proteins (Hu et al., 2003): RL1 is expressed only in pollen, whereas RL2 is expressed ubiquitously, but both are present at very low levels. Deletion of either RL protein causes no detectable defects in root hair development or overall growth (Chen et al., 2011). Whether RL proteins support the role of RHD3 in a tissue-specific manner remains to be investigated.Here, we have analyzed the function of RHD3 and RL proteins in Arabidopsis. We show that RHD3 and the two RL proteins play redundant roles but function during different stages of Arabidopsis development. In addition, we show that RHD3 proteins can functionally replace Sey1p in yeast and mediate ER membrane fusion.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000     

Amino Acid Residue Val362 Plays a Critical Role in Maintaining the Structure of C Terminus of Connexin 50 and in Lens Epithelial-fiber Differentiation

We have previously shown that connexin (Cx) 50, unlike the other two lens connexins, Cx43 and Cx46, promotes chicken lens epithelial-fiber differentiation in a channel-independent manner. Here, we show that deletion of the PEST motif at the C terminus (CT) domain of Cx50 attenuates the stimulatory effect of Cx50 on lens fiber differentiation. Valine 362, a residue located within the PEST domain, is functionally involved. The structure of the Cx50 CT predicted by molecular modeling revealed four α-helices and Val³⁶² was found to be located in the middle of the 3rd helix. Replacement of Val³⁶² with amino acid residues that disrupt the α-helical structure predicted by molecular modeling, such as arginine, glutamate, or phenylalanine, attenuated the stimulatory effects of Cx50 on lens differentiation, whereas replacement with threonine, isoleucine, leucine, or proline, which maintain the structure preserved the function of Cx50. Circular dichroism (CD) studies supported the structural predictions and showed that the substitution with Glu, but not Thr or Pro, disrupted the α-helix, which appears to be the structural feature important for lens epithelial-fiber differentiation. Together, our results suggest that Val³⁶² is important for maintaining the helical structure and is crucial for the role of Cx50 in promoting lens epithelial-fiber differentiation.     

Regulation of the cell expansion gene RHD3 during Arabidopsis development

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.     

Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

FtsK is essential for Escherichia coli cell division. We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with a mukB null mutation resulted in a synthetic lethal phenotype. When the truncated ftsK was combined with a minCDE deletion, chains of minicells were generated, many of which contained DNA. These results suggest that the C terminus of FtsK has an important role in chromosome partitioning.     

Role of the C Terminus of Lassa Virus L Protein in Viral mRNA Synthesis

The N terminus of arenavirus L protein contains an endonuclease presumably involved in “cap snatching.” Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.     

Homotypic Fusion of Immature Secretory Granules during Maturation in a Cell-free Assay

The biogenesis of secretory granules embodies several morphological and biochemical changes. In particular, in neuroendocrine cells maturation of secretory granules is characterized by an increase in size which has been proposed to reflect homotypic fusion of immature secretory granules (ISGs). Here we describe an assay that provides the first biochemical evidence for such a fusion event and allows us to analyze its regulation. The assay reconstitutes homotypic fusion between one population of ISGs containing a [³⁵S]sulfate-labeled substrate, secretogranin II (SgII), and a second population containing the prohormone convertase PC2. Both substrate and enzyme are targeted exclusively to ISGs. Fusion is measured by quantification of a cleavage product of SgII produced by PC2. With this assay we show that fusion only occurs between ISGs and not between ISGs and MSGs, is temperature dependent, and requires ATP and GTP and cytosolic proteins. NSF (N-ethylmaleimide–sensitive fusion protein) is amongst the cytosolic proteins required, whereas we could not detect a requirement for p97. The ability to reconstitute ISG fusion in a cell-free assay is an important advance towards the identification of molecules involved in the maturation of secretory granules and will increase our understanding of this process.     

Regulation of Bestrophin Cl Channels by Calcium: Role of the C Terminus

Human bestrophin-1 (hBest1), which is genetically linked to several kinds of retinopathy and macular degeneration in both humans and dogs, is the founding member of a family of Cl⁻ ion channels that are activated by intracellular Ca²⁺. At present, the structures and mechanisms responsible for Ca²⁺ sensing remain unknown. Here, we have used a combination of molecular modeling, density functional–binding energy calculations, mutagenesis, and patch clamp to identify the regions of hBest1 involved in Ca²⁺ sensing. We identified a cluster of a five contiguous acidic amino acids in the C terminus immediately after the last transmembrane domain, followed by an EF hand and another regulatory domain that are essential for Ca²⁺ sensing by hBest1. The cluster of five amino acids (293–308) is crucial for normal channel gating by Ca²⁺ because all but two of the 35 mutations we made in this region rendered the channel incapable of being activated by Ca²⁺. Using homology models built on the crystal structure of calmodulin (CaM), an EF hand (EF1) was identified in hBest1. EF1 was predicted to bind Ca²⁺ with a slightly higher affinity than the third EF hand of CaM and lower affinity than the second EF hand of troponin C. As predicted by the model, the D312G mutation in the putative Ca²⁺-binding loop (312–323) reduced the apparent Ca²⁺ affinity by 20-fold. In addition, the D312G and D323N mutations abolished Ca²⁺-dependent rundown of the current. Furthermore, analysis of truncation mutants of hBest1 identified a domain adjacent to EF1 that is rich in acidic amino acids (350–390) that is required for Ca²⁺ activation and plays a role in current rundown. These experiments identify a region of hBest1 (312–323) that is involved in the gating of hBest1 by Ca²⁺ and suggest a model in which Ca²⁺ binding to EF1 activates the channel in a process that requires the acidic domain (293–308) and another regulatory domain (350–390). Many of the ~100 disease-causing mutations in hBest1 are located in this region that we have implicated in Ca²⁺ sensing, suggesting that these mutations disrupt hBest1 channel gating by Ca²⁺.     

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.     

拟南芥复制因子C亚基3在黄化苗下胚轴伸长生长中的作用

拟南芥遮光培养2．5d时,rfc3-1突变体黄化幼苗的下胚轴平均长度约比野生型植株黄化幼苗的下胚轴长27．5％。观察表明,相对于野生型复制因子C亚基3（replication factor C3,AtRFC3）基因突变体的下胚轴表皮细胞,特别是上部靠近子叶部分的表皮细胞,单细胞长度变长。将野生型RFC3基因转染到rfc3-1后,突变体恢复野生型表型,进一步说明RFC3在黄化苗的下胚轴伸长生长中有作用。     

Flt3 Does Not Play a Critical Role in Murine Myeloid Leukemias Induced by MLL Fusion Genes

Leukemias harboring MLL translocations are frequent in children and adults, and respond poorly to therapies. The receptor tyrosine kinase FLT3 is highly expressed in these leukemias. In vitro studies have shown that pediatric MLL-rearranged ALL cells are sensitive to FLT3 inhibitors and clinical trials are ongoing to measure their therapeutic efficacy. We sought to determine the contribution of Flt3 in the pathogenesis of MLL-rearranged leukemias using a myeloid leukemia mouse model. Bone marrow from Flt3 null mice transduced with MLL-ENL or MLL-CBP was transplanted into host mice and Flt3 ^−/− leukemias were compared to their Flt3 wild type counterparts. Flt3 deficiency did not delay disease onset and had minimal impact on leukemia characteristics. To determine the anti-leukemic effect of FLT3 inhibition we studied the sensitivity of MLL-ENL leukemia cells to the FLT3 inhibitor PKC412 ex vivo. As previously reported for human MLL-rearranged leukemias, murine MLL-ENL leukemia cells with higher Flt3 levels were more sensitive to the cytotoxicity of PKC412. Interestingly, Flt3 deficient leukemia samples also displayed some sensitivity to PKC412. Our findings demonstrate that myeloid leukemias induced by MLL-rearranged genes are not dependent upon Flt3 signaling. They also highlight the discrepancy between the sensitivity of cells to Flt3 inhibition in vitro and the lack of contribution of Flt3 to the pathogenesis of MLL-rearranged leukemias in vivo.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.     

Juxtamembranous Region of the Amino Terminus of the Family B G Protein-coupled Calcitonin Receptor Plays a Critical Role in Small-molecule Agonist Action

The Family B G protein-coupled calcitonin receptor is an important drug target. The aim of this work was to elucidate the molecular mechanism of action of small-molecule agonist ligands acting at this receptor, comparing it with the action mechanism of the receptor''s natural peptide ligand. cAMP responses to four non-peptidyl ligands and calcitonin were studied in COS-1 cells expressing wild-type and chimeric calcitonin-secretin receptors. All compounds were full agonists at the calcitonin receptor with no activity at the secretin receptor. Only chimeric constructs including the calcitonin receptor amino terminus exhibited responses to any of these ligands. We progressively truncated this domain and tested constructs for cAMP responses. Although calcitonin was able to activate the calcitonin receptor fully with the first 58 residues absent, its potency was 3 orders of magnitude lower than that at the wild-type receptor. After truncation of 114 residues, there was no response to calcitonin. In contrast, small-molecule ligands were fully active at receptors having up to 149 amino-terminal residues absent. Those compounds finally became inactive after truncation of 153 residues. Deletion and/or alanine replacement of the region of the calcitonin receptor between residues 150 and 153 resulted in marked reduction in cAMP responses to these compounds, with some compound-specific differences observed, supporting a critical role for this region. Binding studies further supported distinct sites of action of small molecules relative to that of calcitonin. These findings focus attention on the potential importance of the juxtamembranous region of the amino terminus of the Family B calcitonin receptor for agonist drug action.Calcitonin (CT),³ a 32-amino acid peptide secreted by the thyroid gland in response to elevations in blood calcium levels, acts on bone and kidney to maintain calcium homeostasis (1, 2). CT is widely used therapeutically for the treatment of bone-related disorders such as osteoporosis, hypercalcemia of malignancy, and Paget disease (1, 2). This peptide must be administered parenterally, whereas small-molecule agonist ligands that can be administered orally have substantial clinical advantage, particularly for long-term treatment.CT acts on the CT receptor, a Family B G protein-coupled receptor (GPCR). Members of this family include receptors for secretin, vasoactive intestinal polypeptide, parathyroid hormone, corticotrophin-releasing factor, glucagon, glucagon-like peptide 1, CT, and CT gene-related peptide (3), with each having a long extracellular amino-terminal domain containing six conserved cysteine residues that form three intradomain disulfide bonds. This region has been shown to play a critical role in natural peptide ligand binding and receptor activation (4–11).Development of small-molecule ligands for Family B GPCRs represents an area of great interest. For the CT receptor, several such ligands have been developed (12–14). However, the mechanisms of their actions at their receptors remain unclear. Most recently, synthesis of a series of pyrazolopyridine CT receptor ligands described as partial agonists has been reported (15). Here, we explore the structural basis for their action at the CT receptor. Using CT-secretin receptor chimeric analysis, the amino terminus of the CT receptor was identified as a critical region for the action of these small-molecule ligands but with determinants that are distinct from those interacting with the natural ligand, CT. Truncation, deletion, and alanine replacement mutations of the CT receptor identified that three specific receptor residues, Tyr¹⁵⁰, Leu¹⁵¹, and Ile¹⁵³, within the juxtamembranous region of the amino-terminal domain of the CT receptor, play important roles in the actions of these small-molecule agonists. This study represents the first identification of this region as being critical for the action of small-molecule drugs acting at Family B GPCRs.     

The Role of the Target Membrane Structure in Fusion with Sendai Virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 ?C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

Cysteine 195 Has a Critical Functional Role in Catalysis by Isocitrate Lyase from Escherichia coli

Cysteine 195 in isocitrate lyase from Escherichia coli has been replaced by directed mutagenesis. Substitution by Ser yields enzyme with a k_cat that is 0.03% that of wild type, and substitution by Ala, Gly, Thr, or Val yields completely inactive enzyme. The present results are consistent with a functional role of Cys 195. Received: 26 March 1997 / Accepted: 29 April 1997