scholarly journals The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties

1994 ◽  
Vol 107 (4) ◽  
pp. 1019-1029 ◽  
Author(s):  
H. Hennekes ◽  
E.A. Nigg
Keyword(s):  
Mammalian Cells ◽  
Membrane Binding ◽  
Proteolytic Cleavage ◽  
Single Point Mutation ◽  
Lamin A ◽  
Binding Properties ◽  
Nuclear Lamins ◽  
C Terminus ◽  
Membrane Attachment

Mature A- and B-type lamins differ in the extent to which they interact with the nuclear membrane and thus represent an interesting model for studying the role of isoprenylation and carboxyl-methylation in membrane attachment. Both A- and B-type lamins are isoprenylated and carboxyl-methylated shortly after synthesis, but A-type lamins undergo a further proteolytic cleavage which results in the loss of the hydrophobically modified C terminus. Here, we have constructed mutants of chicken lamin A that differ in their abilities to serve as substrates for different post-translational processing events occurring at the C terminus of the wild-type precursor. In addition to studying full-length proteins, we have analyzed C-terminal end domains of lamin A, either alone or after fusion to reporter proteins. Mutant proteins were expressed in mammalian cells, and their membrane association was analyzed by immunofluorescence microscopy and subcellular fractionation. Our results provide information on the substrate specificity and subcellular localization of the lamin-A-specific protease. Moreover, they indicate that hydrophobic modifications of the C-terminal end domains account for the differential membrane-binding properties of A- and B-type lamins. Thus, some of the integral membrane proteins implicated in anchoring B-type lamins to the membrane may function as receptors for the isoprenylated and carboxyl-methylated C terminus.

Role of Cationic Groove and Hydrophobic Residues in Phosphatidylinositol-Dependent Membrane-Binding Properties of Tks5-Phox Homology Domain

2018 ◽  
Vol 3 (4) ◽  
pp. 1205-1214 ◽  
Author(s):  
Sukhamoy Gorai ◽  
Debasish Paul ◽  
Rituparna Borah ◽  
Nandan Haloi ◽  
Manas Kumar Santra ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Binding Properties ◽  
Hydrophobic Residues ◽  
Phox Homology

Abstract 52: The Role of Kindlin-2 in Integrin Activation Depends on a Short C-Terminal Region

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Jamila Hirbawi ◽  
Kamila Bledzka ◽  
Yan Qing Ma ◽  
Jun Qin ◽  
Edward F Plow
Keyword(s):  
Amino Acids ◽  
Point Mutation ◽  
Single Point ◽  
Terminal Region ◽  
Membrane Receptors ◽  
Single Point Mutation ◽  
Loss Of Function ◽  
Integrin Activation ◽  
C Terminus

Integrins are heterodimeric cell membrane receptors that regulate cell adhesion, migration, and survival. The kindlins are known to be key regulators of integrin activation, the transition from a low affinity, default state to a high affinity state for ligand. This function depends on their binding, together with talin, to the cytoplasmic tails (CT) of the β subunit of integrins. Kindlins are FERM domain containing proteins, and it is its F3 (PTB) subdomain of the FERM that is the primary binding site for integrin β CT. At its very C-terminus, beyond the F3, is a short extension of 21 amino acids, K2 660-680, and we have focused on the role of this region in the co-activator function of kindlin-2 (K2). For this analysis, we performed PAC-1 (antibody to detect activated αIIbβ3 integrin) binding assays in CHO cells stably expressing integrin α IIb β 3 that were transiently transfected with talin head domain and K2 mutants. Expression levels of all proteins were verified to be similar by western blotting and FACS. Truncation of K2 at residue 660 essentially eliminated the co-activator function of K2. Deletion of smaller segments also reduced co-activator activity by 50% to 100%. Deletion of just the last two amino acids in the sequence, W 679 V 680 , resulted in a 50% reduction in co-activator activity and a single point mutation of Y 673 A also led to a 50% loss of function. A combination mutant consisting of the W 679 V 680 deletion and the Y 673 point mutation resulted in 100% loss of kindlin-2 co-activator activity. Pull-down experiments performed using GST tagged β 3 CT and CHO lysates transfected with GFP-kindlin-2 forms suggested that the C-terminal deletion did not disrupt binding to β 3 CT. This observation was corroborated by surface plasmon resonance studies in which the binding of full-length K2 and K2Δ666C (Δ666) was compared, and their K D values for immobilized β3 CT were found to be essentially the same. Overall, these data establish an important and unanticipated role of the carboxy-terminal region of kindlin-2 in its integrin co-activator function that is not dependent of its binding to integrin.

scholarly journals Cell cycle–regulated membrane binding of NuMA contributes to efficient anaphase chromosome separation

2014 ◽  
Vol 25 (5) ◽  
pp. 606-619 ◽  
Author(s):  
Zhen Zheng ◽  
Qingwen Wan ◽  
Gerry Meixiong ◽  
Quansheng Du
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Membrane Binding ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Α Subunit ◽  
Definitive Evidence ◽  
C Terminus ◽  
Chromosome Separation ◽  
Spindle Elongation

Accurate and efficient separation of sister chromatids during anaphase is critical for faithful cell division. It has been proposed that cortical dynein–generated pulling forces on astral microtubules contribute to anaphase spindle elongation and chromosome separation. In mammalian cells, however, definitive evidence for the involvement of cortical dynein in chromosome separation is missing. It is believed that dynein is recruited and anchored at the cell cortex during mitosis by the α subunit of heterotrimeric G protein (Gα)/mammalian homologue of Drosophila Partner of Inscuteable/nuclear mitotic apparatus (NuMA) ternary complex. Here we uncover a Gα/LGN-independent lipid- and membrane-binding domain at the C-terminus of NuMA. We show that the membrane binding of NuMA is cell cycle regulated—it is inhibited during prophase and metaphase by cyclin-dependent kinase 1 (CDK1)–mediated phosphorylation and only occurs after anaphase onset when CDK1 activity is down-regulated. Further studies indicate that cell cycle–regulated membrane association of NuMA underlies anaphase-specific enhancement of cortical NuMA and dynein. By replacing endogenous NuMA with membrane-binding-deficient NuMA, we can specifically reduce the cortical accumulation of NuMA and dynein during anaphase and demonstrate that cortical NuMA and dynein contribute to efficient chromosome separation in mammalian cells.

Expression of nuclear lamins during mouse preimplantation development

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 271-278
Author(s):  
E. Houliston ◽  
M.N. Guilly ◽  
J.C. Courvalin ◽  
B. Maro
Keyword(s):  
Cell Differentiation ◽  
Early Development ◽  
Nuclear Periphery ◽  
Preimplantation Development ◽  
Lamin A ◽  
Positive Staining ◽  
Lamin B ◽  
Nuclear Lamins ◽  
Mouse Cells

The expression of nuclear lamins during mouse preimplantation development was studied by immunofluorescence, immunoblotting and immunoprecipitation. Two sera were used, specific either for lamin B or lamins A and C. Both sera gave a positive staining of the nuclear periphery throughout preimplantation development (fertilized eggs to late blastocysts). Immunoblots revealed that the three lamins were present in eggs and blastocysts. However, lamin A from eggs was found to have a higher apparent Mr than lamin A from blastocysts and other mouse cells. Using immunoprecipitation, synthesis of lamin A was detected in eggs while synthesis of lamin B was detected in 8-cell embryos and blastocysts, indicating that at least some of the lamins used during early development do not come from a store in the egg. These results are discussed in relation to the possible role of lamins during cell differentiation.

scholarly journals Role of the FtsA C Terminus as a Switch for Polymerization and Membrane Association

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Marcin Krupka ◽  
Elisa J. Cabré ◽  
Mercedes Jiménez ◽  
Germán Rivas ◽  
Ana Isabel Rico ◽  
...  
Keyword(s):  
Membrane Vesicles ◽  
Lipid Monolayers ◽  
Membrane Association ◽  
Amphipathic Helix ◽  
Content Type ◽  
Ftsz Ring ◽  
C Terminus ◽  
Membrane Attachment ◽  
Attachment Devices

ABSTRACT Together with ATP, the C-terminal region of the essential streptococcal FtsA protein acts as an intramolecular switch to promote its polymerization and attachment to the membrane. During septation, FtsA is known to anchor the constricting FtsZ ring and, subsequently, the divisome to the membrane. Truncation of the C terminus of the streptococcal FtsA (FtsAΔCt) facilitates a more rapid ATP-dependent polymerization in solution than is seen with the full-length protein (FtsA+). The FtsAΔCt polymers are more organized and compact than those formed in solution by FtsA+, resembling the shape of the membrane-associated FtsA+ polymers. We find that ATP, besides being needed for polymerization, is required for the attachment of FtsA+ to lipid monolayers and to vesicle membranes. We propose a model in which the binding of ATP activates a switch favoring the polymerization of FtsA and at the same time driving the amphipathic helix at its C terminus to become attached to the membrane. Conversely, when FtsA is in the cytoplasm, the C terminus is not engaged in the attachment to the membrane, and it obstructs polymerization. ATP-dependent polymerization of FtsA inside membrane vesicles causes vesicle shrinkage, suggesting that, besides providing a membrane attachment for FtsZ, the FtsA C terminus may also introduce local alterations in the membrane to facilitate septation. IMPORTANCE FtsA is a protein needed in many bacteria to construct a septum that divides one fully grown cell, producing two daughters. We show that the region located at the C-terminal end of the Streptococcus pneumoniae FtsA protein works as a switch triggered by ATP, a molecule that stores energy. This region contains an amphipathic helix that obstructs the assembly of FtsA into polymers in the cytoplasm. In the presence of ATP, the obstruction is removed by switching the position of the helix. The switch directs the helix to the membrane and simultaneously facilitates the polymerization of the protein. The accumulation of FtsA molecules at the membrane causes distortions, an effect produced also by proteins such as MinD, MreB, and SepF that also contain amphipathic helixes as membrane attachment devices. In the case of FtsA, these distortions may also facilitate the initial events that lead to the division of bacteria.

scholarly journals Role of the C Terminus of Foamy Virus Gag in RNA Packaging and Pol Expression

2004 ◽  
Vol 78 (17) ◽  
pp. 9423-9430 ◽  
Author(s):  
Carolyn R. Stenbak ◽  
Maxine L. Linial
Keyword(s):  
Foamy Virus ◽  
Nucleic Acid Binding ◽  
Rna Packaging ◽  
Binding Properties ◽  
Gag Protein ◽  
C Terminus ◽  
Foamy Viruses ◽  
The Matrix

ABSTRACT Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol proteins are expressed independently of one another, and both proteins undergo single cleavage events. Thus, the mature FV Gag protein does not consist of the matrix, capsid, and nucleocapsid (NC) proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol protein.

scholarly journals The Golgi-associated Protein GRASP65 Regulates Spindle Dynamics and Is Essential for Cell Division

2005 ◽  
Vol 16 (7) ◽  
pp. 3211-3222 ◽  
Author(s):  
Christine Sütterlin ◽  
Roman Polishchuk ◽  
Matt Pecot ◽  
Vivek Malhotra
Keyword(s):  
Mammalian Cells ◽  
Protein Transport ◽  
Direct Role ◽  
Golgi Stack ◽  
Spindle Poles ◽  
C Terminus ◽  
Spindle Dynamics ◽  
Negative Role ◽  
Golgi Fragmentation

At the onset of mitosis, the pericentriolar Golgi apparatus of mammalian cells is converted into small fragments, which are dispersed throughout the cytosol. The Golgi-associated protein GRASP65 is involved in this process. To address the role of GRASP65 in mitotic Golgi fragmentation, we depleted the protein from HeLa cells by RNAi. In the absence of GRASP65, the number of cisternae per Golgi stack is reduced without affecting the overall organization of Golgi membranes and protein transport. GRASP65-depleted cells entered mitosis, but accumulated in metaphase with condensed chromatin and multiple aberrant spindles and eventually died. Although Centrin2 and g-tubulin were detected in two of the spindle poles, the other spindle poles contained g-tubulin, but not Centrin2. Furthermore, we provide evidence that the expression of the C-terminus of GRASP65 interferes with entry of cells into mitosis. Our results suggest the requirement for GRASP65 in the regulation of spindle dynamics rather than a direct role in the stacking of Golgi cisternae. This novel function is in addition to the previously established negative role of GRASP65 at the G2/M transition, which is mediated by its C-terminus.

The role of sequences unique to nuclear intermediate filaments in the targeting and assembly of human lamin B: evidence for lack of interaction of lamin B with its putative receptor

1998 ◽  
Vol 111 (23) ◽  
pp. 3471-3485 ◽  
Author(s):  
T.I. Mical ◽  
M.J. Monteiro
Keyword(s):  
Mammalian Cells ◽  
Nuclear Lamina ◽  
Lamin B ◽  
Nuclear Lamins ◽  
Putative Receptor ◽  
Lamin B Receptor ◽  
Nuclear Lamin ◽  
Efficient Integration ◽  
Caax Motif

The mechanism by which human nuclear lamin B is targeted and assembled has been studied by transfecting into mammalian cells lamin mutants deleted of three sequences unique to lamins. Nuclear lamins contain an extra 42 amino acids (aa) in their rod domains and NLS and CAAX motifs in their tail domains, which distinguishes them from cytoplasmic IF proteins. These three sequences act in concert to ensure correct temporal and spatial assembly of lamin B. Deletion of any one of these three sequences from lamin B did not significantly disrupt nuclear lamina targeting, but when two or more of these sequences were deleted, targeting was severely compromised. The CAAX motif is necessary for the efficient integration of lamin B into an already formed nuclear lamina, since lamin B CAAX- mutants had reduced targeting to the lamina when arrested in S phase of the cell cycle. CAAX-deficient mutant lamin B proteins were soluble and not associated with membranes at mitosis, proving that the CAAX motif is responsible for association of human lamin B with membranes. In addition, CAAX- mutant lamin B proteins fractionated independently of the lamin B-receptor (LBR), indicating that these two proteins do not bind directly to each other.

Selective proteolytic cleavage of recombinant human interleukin 4. Evidence for a critical role of the C-terminus

Biochemistry ◽  
1991 ◽  
Vol 30 (40) ◽  
pp. 9576-9582 ◽  
Author(s):  
Hung V. Le ◽  
Gail F. Seelig ◽  
Rosalinda Syto ◽  
Lata Ramanathan ◽  
William T. Windsor ◽  
...  
Keyword(s):  
Critical Role ◽  
Proteolytic Cleavage ◽  
Interleukin 4 ◽  
C Terminus

scholarly journals Identification of essential genes in cultured mammalian cells using small interfering RNAs

2001 ◽  
Vol 114 (24) ◽  
pp. 4557-4565 ◽  
Author(s):  
Jens Harborth ◽  
Sayda M. Elbashir ◽  
Kim Bechert ◽  
Thomas Tuschl ◽  
Klaus Weber
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Cytoplasmic Dynein ◽  
Essential Genes ◽  
Lamin A ◽  
Small Interfering Rnas ◽  
Nuclear Lamins ◽  
Culture Cells ◽  
Altered Cell ◽  
Nonessential Genes

We report the first RNAi-induced phenotypes in mammalian cultured cells using RNA interference mediated by duplexes of 21-nt RNAs. The 21 gene products studied have different functions and subcellular localizations. Knockdown experiments monitored by immunofluorescence and immunoblotting show that even major cellular proteins such as actin and vimentin can be silenced efficiently. Genes were classified as essential or nonessential depending on impaired cell growth after RNA silencing. Phenotypes also involved altered cell morphology and aberrant mitotic arrest. Among the essential genes identified by RNAi for which such information was previously not available are lamin B1, lamin B2, NUP153, GAS41, ARC21, cytoplasmic dynein, the protein kinase cdk1 and both β- and γ-actin. Newly defined nonessential genes are emerin and zyxin. Several genes previously characterized by other methods such as knockout of murine genes are included as internal controls and gave identical results when RNAi was used. In the case of two nonessential genes (lamin A/C and zyxin) RNAi provides a recognizable phenotype. Our results complete the characterization of the mammalian nuclear lamins. While lamins A/C appear as nonessential proteins in the mouse embryo and in RNAi treated cultured cells, the two other lamins, B1 and B2, are now identified as essential proteins. Interestingly the inner nuclear membrane protein emerin, thought to be a ligand of lamin A/C, is also a nonessential protein in tissue culture cells.

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