Dynamin-Like Proteins Are Potentially Involved in Membrane Dynamics within Chloroplasts and Cyanobacteria.
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ABSTRACT:
SUBMITTER: Jilly R
PROVIDER: S-EPMC5827413 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
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Project description:Membrane proteins that are imported into chloroplasts must be accurately targeted in order to maintain the identity and function of the highly differentiated internal membranes. Relatively little is known about the targeting information or pathways that direct proteins with transmembrane domains to either the inner envelope or thylakoids. In this study, we focused on a structurally simple class of membrane proteins, the tail-anchored proteins, which have stroma-exposed amino-terminal domains and a single transmembrane domain within 30 amino acids of the carboxy-terminus. SECE1 and SECE2 are essential tail-anchored proteins that function as components of the dual SEC translocases in chloroplasts. SECE1 localizes to the thylakoids, while SECE2 localizes to the inner envelope. We have used transient expression in Arabidopsis leaf protoplasts and confocal microscopy in combination with a domain-swapping strategy to identify regions that contain important targeting determinants. We show that membrane-specific targeting depends on features of the transmembrane domains and the short C-terminal tails. We probed the contributions of these regions to targeting processes further through site-directed mutagenesis. We show that thylakoid targeting still occurs when changes are made to the tail of SECE1, but changing residues in the tail of SECE2 abolishes inner envelope targeting. Finally, we discuss possible parallels between sorting of tail-anchored proteins in the stroma and in the cytosol.
| S-EPMC6857650 | biostudies-literature
Project description:Dynamin superfamily molecular motors use guanosine triphosphate (GTP) as a source of energy for membrane-remodeling events. We found that knockdown of nucleoside diphosphate kinases (NDPKs) NM23-H1/H2, which produce GTP through adenosine triphosphate (ATP)-driven conversion of guanosine diphosphate (GDP), inhibited dynamin-mediated endocytosis. NM23-H1/H2 localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin. In vitro, NM23-H1/H2 were recruited to dynamin-induced tubules, stimulated GTP-loading on dynamin, and triggered fission in the presence of ATP and GDP. NM23-H4, a mitochondria-specific NDPK, colocalized with mitochondrial dynamin-like OPA1 involved in mitochondria inner membrane fusion and increased GTP-loading on OPA1. Like OPA1 loss of function, silencing of NM23-H4 but not NM23-H1/H2 resulted in mitochondrial fragmentation, reflecting fusion defects. Thus, NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to work with high thermodynamic efficiency.
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Project description:Cells have evolved diverse protein-based machinery to reshape, cut, or fuse their membrane-delimited compartments. Dynamin superfamily proteins are principal components of this machinery and use their ability to hydrolyze GTP and to polymerize into helices and rings to achieve these goals. Nucleotide-binding, hydrolysis, and exchange reactions drive significant conformational changes across the dynamin family, and these changes alter the shape and stability of supramolecular dynamin oligomers, as well as the ability of dynamins to bind receptors and membranes. Mutations that interfere with the conformational repertoire of these enzymes, and hence with membrane fission, exist in several inherited human diseases. Here, we discuss insights from new x-ray crystal structures and cryo-EM reconstructions that have enabled us to infer some of the allosteric dynamics for these proteins. Together, these studies help us to understand how dynamins perform mechanical work, as well as how specific mutants of dynamin family proteins exhibit pathogenic properties.
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Project description:BackgroundMitochondrial morphology is maintained by two distinct membrane events -fission and fusion. Altering these conserved processes can disrupt mitochondrial morphology and distribution, thereby disrupting the organelle's functionality and impeding cellular function. In higher eukaryotes, these processes are mediated by a family of dynamin-related proteins (DRP's). In the lower eukaryotes, for instance Dictyostelium discoideum, mitochondrial fission and fusion have been implicated but not yet established. To understand the overall mechanism of these dynamics across organisms, we developed an assay to identify fission and fusion events in Dictyostelium and to assess the involvement of the mitochondrial proteins, MidA, CluA, and two DRP's, DymA and DymB.FindingsUsing laser scanning confocal microscopy we show, for the first time, that lower eukaryotes mediate mitochondrial fission and fusion. In Dictyostelium, these processes are balanced, occurring approximately 1 event/minute. Quantification of the rates in midA-, cluA-, dymA-, or dymB- strains established that MidA appears to play an indirect role in the regulation of fission and fusion, while the DRP's are not essential for these processes. Rates of fission and fusion were significantly reduced in cluA-cells, indicating that CluA is necessary for maintaining both fission and fusion.ConclusionsWe have successfully demonstrated that Dictyostelium mitochondria undergo the dynamic processes of fission and fusion. The classical mediators of membrane dynamics - the DRP's - are not necessary for these dynamics, whereas CluA is necessary for both processes. This work contributes to our overall understanding of mitochondrial dynamics and ultimately will provide additional insight into mitochondrial disease.
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Project description:PurposeSpatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (βPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying βPix-mediated cancer cell invasion remain unclear.MethodsThe clinical significance of βPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for βPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of βPix on CRC progression. A βPix-SH3 antibody delivery system was used to determine the effects of the βPix-Dyn2 complex in CRC cells.ResultsWe found that the Src homology 3 (SH3) domain of βPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The βPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of βPix enhanced βPix-Dyn2 complex formation. Disruption of the βPix-Dyn2 complex by βPix-SH3 antibodies targeting intracellular βPix inhibited CRC cell invasion.ConclusionsOur data indicate that spatiotemporal regulation of the Src-βPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the βPix-Dyn2 complex may be a useful therapeutic strategy for CRC.
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Project description:The IM30 (inner membrane-associated protein of 30 kDa), also known as the Vipp1 (vesicle-inducing protein in plastids 1), has a crucial role in thylakoid membrane biogenesis and maintenance. Recent results suggest that the protein binds peripherally to membranes containing negatively charged lipids. However, although IM30 monomers interact and assemble into large oligomeric ring complexes with different numbers of monomers, it is still an open question whether ring formation is crucial for membrane interaction. Here we show that binding of IM30 rings to negatively charged phosphatidylglycerol membrane surfaces results in a higher ordered membrane state, both in the head group and in the inner core region of the lipid bilayer. Furthermore, by using gold nanorods covered with phosphatidylglycerol layers and single particle spectroscopy, we show that not only IM30 rings but also lower oligomeric IM30 structures interact with membranes, although with higher affinity. Thus, ring formation is not crucial for, and even counteracts, membrane interaction of IM30.
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Project description:Peripheral membrane proteins with intrinsic curvature can act both as sensors of membrane curvature and shape modulators of the underlying membranes. A well-studied example of such proteins is the mechanochemical GTPase dynamin, which assembles into helical filaments around membrane tubes and catalyzes their scission in a GTPase-dependent manner. It is known that the dynamin coat alone, without GTP, can constrict membrane tubes to radii of ?10 nm, indicating that the intrinsic shape and elasticity of dynamin filaments should play an important role in membrane remodeling. However, molecular and dynamic understanding of the process is lacking. Here, we develop a dynamical polymer-chain model for a helical elastic filament bound on a deformable membrane tube of conserved mass, accounting for thermal fluctuations in the filament and lipid flows in the membrane. The model is based on the locally cylindrical helix approximation for dynamin. We obtain the elastic parameters of the dynamin filament by molecular dynamics simulations of its tetrameric building block and also from coarse-grained structure-based simulations of a 17-dimer filament. The results show that the stiffness of dynamin is comparable to that of the membrane. We determine equilibrium shapes of the filament and the membrane and find that mostly the pitch of the filament, not its radius, is sensitive to variations in membrane tension and stiffness. The close correspondence between experimental estimates of the inner tube radius and those predicted by the model suggests that dynamin's "stalk" region is responsible for its GTP-independent membrane-shaping ability. The model paves the way for future mesoscopic modeling of dynamin with explicit motor function.
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Project description:A recent model for peroxisome biogenesis postulates that peroxisomes form de novo continuously in wild-type cells by heterotypic fusion of endoplasmic reticulum-derived vesicles containing distinct sets of peroxisomal membrane proteins. This model proposes a role in vesicle fusion for the Pex1/Pex6 complex, which has an established role in matrix protein import. The growth and division model proposes that peroxisomes derive from existing peroxisomes. We tested these models by reexamining the role of Pex1/Pex6 and dynamin-related proteins in peroxisome biogenesis. We found that induced depletion of Pex1 blocks the import of matrix proteins but does not affect membrane protein delivery to peroxisomes; markers for the previously reported distinct vesicles colocalize in pex1 and pex6 cells; peroxisomes undergo continued growth if fission is blocked. Our data are compatible with the established primary role of the Pex1/Pex6 complex in matrix protein import and show that peroxisomes in Saccharomyces cerevisiae multiply mainly by growth and division.
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Project description:The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds' necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations ( approximately 0.1-10 microM), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration.
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