scholarly journals Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities

2016 ◽  
Vol 213 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Holly A. Ping ◽  
Lauren M. Kraft ◽  
WeiTing Chen ◽  
Amy E. Nilles ◽  
Laura L. Lackner
Keyword(s):  
Plasma Membrane ◽  
Specific Binding ◽  
Coiled Coil ◽  
Lipid Binding ◽  
General Mechanism ◽  
Cell Cortex ◽  
Pleckstrin Homology Domain ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Mitochondrial Distribution

The mitochondria–ER cortex anchor (MECA) is required for proper mitochondrial distribution and functions by tethering mitochondria to the plasma membrane. The core component of MECA is the multidomain protein Num1, which assembles into clusters at the cell cortex. We show Num1 adopts an extended, polarized conformation. Its N-terminal coiled-coil domain (Num1CC) is proximal to mitochondria, and the C-terminal pleckstrin homology domain is associated with the plasma membrane. We find that Num1CC interacts directly with phospholipid membranes and displays a strong preference for the mitochondria-specific phospholipid cardiolipin. This direct membrane interaction is critical for MECA function. Thus, mitochondrial anchoring is mediated by a protein that interacts directly with two different membranes through lipid-specific binding domains, suggesting a general mechanism for interorganelle tethering.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes

2015 ◽  
Vol 309 (12) ◽  
pp. H2127-H2136 ◽  
Author(s):  
Daisuke Naito ◽  
Takehiro Ogata ◽  
Tetsuro Hamaoka ◽  
Naohiko Nakanishi ◽  
Kotaro Miyagawa ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cardiac Function ◽  
Membrane Trafficking ◽  
Protein Level ◽  
Interstitial Fibrosis ◽  
Coiled Coil ◽  
Cardiomyocyte Hypertrophy ◽  
Coiled Coil Domain ◽  
Cytoplasmic Proteins ◽  
And Function

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

scholarly journals Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

2012 ◽  
Vol 109 (38) ◽  
pp. E2533-E2542 ◽  
Author(s):  
Michal Skruzny ◽  
Thorsten Brach ◽  
Rodolfo Ciuffa ◽  
Sofia Rybina ◽  
Malte Wachsmuth ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Lipid Binding ◽  
Actin Binding ◽  
Dependent Manner ◽  
Vesicle Budding ◽  
Cooperative Action ◽  
Binding Domains ◽  
Membrane Invagination

Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane–actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2’s ANTH and Ent1’s ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.

scholarly journals Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs

2016 ◽  
Vol 113 (50) ◽  
pp. E8069-E8078 ◽  
Author(s):  
Miriam Stoeber ◽  
Pascale Schellenberger ◽  
C. Alistair Siebert ◽  
Cedric Leyrat ◽  
Ari Helenius ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Complex Formation ◽  
Membrane Binding ◽  
Coiled Coil ◽  
Structural Basis ◽  
Membrane Domains ◽  
Coiled Coil Domain ◽  
Regular Dodecahedron ◽  
Plasma Membrane Domains ◽  
Electron Cryotomography

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.

scholarly journals Structure of full-length human TRPM4

2018 ◽  
Vol 115 (10) ◽  
pp. 2377-2382 ◽  
Author(s):  
Jingjing Duan ◽  
Zongli Li ◽  
Jian Li ◽  
Ana Santa-Cruz ◽  
Silvia Sanchez-Martinez ◽  
...  
Keyword(s):  
Transient Receptor Potential ◽  
Trp Channels ◽  
Coiled Coil ◽  
Receptor Potential ◽  
Lipid Binding ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Coiled Coil Domain ◽  
Transient Receptor Potential Melastatin ◽  
Transient Receptor

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na+-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4–S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π–π bonds and cation–π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

Fa1p is a 171 kDa protein essential for axonemal microtubule severing in Chlamydomonas

2000 ◽  
Vol 113 (11) ◽  
pp. 1963-1971 ◽  
Author(s):  
R.J. Finst ◽  
P.J. Kim ◽  
E.R. Griffis ◽  
L.M. Quarmby
Keyword(s):  
Coiled Coil ◽  
Subcellular Fractionation ◽  
Rt Pcr ◽  
Type Locus ◽  
Microtubule Severing ◽  
Calmodulin Binding ◽  
Amino Terminal ◽  
Binding Domains ◽  
Coiled Coil Domain ◽  
Novel Protein

A key event in deflagellation or deciliation is the severing of the nine outer-doublet axonemal microtubules at a specific site in the flagellar transition zone. Previous genetic analysis revealed three genes that are essential for deflagellation in Chlamydomonas. We have now identified the first of these products, Fa1p, a protein required for Ca(2+)-dependent, axonemal microtubule severing. Genetic mapping and the availability of a tagged allele allowed us to physically map the gene to the centromere-proximal domain of the mating-type locus. We identified clones of Chlamydomonas genomic DNA that rescued the Ca(2+)-dependent axonemal microtubule severing defect of fa1 mutants. The FA1 cDNA, obtained by RT-PCR, encodes a novel protein of 171 kDa, which is predicted to contain an amino-terminal coiled-coil domain and three Ca(2+)/calmodulin binding domains. By western analysis and subcellular fractionation, the FA1 product is enriched in flagellar-basal body complexes. Based on these observations and previous studies, we hypothesize that a Ca(2+)-activated, Ca(2+)-binding protein binds Fa1p leading ultimately to the activation of axonemal microtubule severing.

scholarly journals Nucleolar Trafficking of Nucleostemin Family Proteins: Common versus Protein-Specific Mechanisms

2007 ◽  
Vol 27 (24) ◽  
pp. 8670-8682 ◽  
Author(s):  
Lingjun Meng ◽  
Qubo Zhu ◽  
Robert Y. L. Tsai
Keyword(s):  
Coiled Coil ◽  
Dependent Manner ◽  
Guanine Nucleotide Binding Protein ◽  
Binding Domains ◽  
Gtp Binding ◽  
Coiled Coil Domain ◽  
Versus Protein ◽  
Localization Signal ◽  
Guanine Nucleotide Binding ◽  
Retention Signal

ABSTRACT The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins.

scholarly journals Phase separation of Ede1 promotes the initiation of endocytic events

2019 ◽  
Author(s):  
Mateusz Kozak ◽  
Marko Kaksonen
Keyword(s):  
Plasma Membrane ◽  
Phase Separation ◽  
Coiled Coil ◽  
Dynamic Network ◽  
Lipid Binding ◽  
Core Region ◽  
Initiation Factor ◽  
Major Pathway ◽  
The Core ◽  
Endocytic Proteins

AbstractClathrin-mediated endocytosis is a major pathway that eukaryotic cells use to produce transport vesicles from the plasma membrane. The assembly of the endocytic coat is initiated by a dynamic network of weakly interacting proteins, but the exact mechanism of initiation is unknown. Ede1, the yeast homologue of mammalian Eps15, is one of the early-arriving endocytic proteins and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and the frequency of endocytic initiation is decreased. We show here that in mutants with increased amounts of cytoplasmic Ede1, the excess protein forms large condensates which exhibit properties of phase separated liquid protein droplets. These Ede1 condensates recruit many other early-arriving endocytic proteins. Their formation depends on the core region of Ede1 that contains a coiled coil and a low-complexity domain. We demonstrate that Ede1 core region is essential for the endocytic function of Ede1. The core region can also promote clustering of a heterologous lipid-binding domain into discrete sites on the plasma membrane that initiate endocytic events. We propose that the clustering of the early endocytic proteins and cargo depend on phase separation mediated by Ede1.

scholarly journals Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

2007 ◽  
Vol 18 (12) ◽  
pp. 4945-4956 ◽  
Author(s):  
Satoe Takahashi ◽  
Peter M. Pryciak
Keyword(s):  
Map Kinase ◽  
Fluorescent Protein ◽  
Membrane Binding ◽  
Eukaryotic Cell ◽  
Lipid Binding ◽  
Cell Cortex ◽  
Membrane Phospholipids ◽  
Binding Domains

The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP2-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase–dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein–protein and protein–membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex.

scholarly journals Role of the EF-hand and coiled-coil domains of human Rab44 in localisation and organelle formation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kohei Ogawa ◽  
Tomoko Kadowaki ◽  
Mitsuko Tokuhisa ◽  
Yu Yamaguchi ◽  
Masahiro Umeda ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coiled Coil ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Rab Gtpase ◽  
Amino Terminal ◽  
Coiled Coil Domain ◽  
Ef Hand ◽  
Carboxyl Terminal

Abstract Rab44 is a large Rab GTPase that contains an amino-terminal EF-hand domain, a coiled-coil domain, and a carboxyl-terminal Rab GTPase domain. However, the roles of the EF-hand and coiled-coil domains remain unclear. Here, we constructed various deletion and point mutants of human Rab44. When overexpressed in HeLa cells, the wild-type Rab44 (hWT) formed ring-like structures, and partially localised to lysosomes. The dominant negative mutant, hT847N, localised to lysosomes and the cytosol, while the constitutively active mutant, hQ892L, formed ring-like structures, and partially localised to the plasma membrane and nuclei. The hΔEF, hΔcoil, and h826-1021 mutants also formed ring-like structures; however, their localisation patterns differed from hWT. Analysis of live imaging with LysoTracker revealed that the size of LysoTracker-positive vesicles was altered by all other mutations than the hC1019A and hΔEF. Treatment with ionomycin, a Ca2+ ionophore, induced the translocation of hWT and hΔcoil into the plasma membrane and cytosol, but had no effect on the localisation of the hΔEF and h826-1021 mutants. Thus, the EF- hand domain is likely required for the partial translocation of Rab44 to the plasma membrane and cytosol following transient Ca2+ influx, and the coiled-coil domain appears to be important for localisation and organelle formation.

Sign in / Sign up

Export Citation Format

Share Document