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Results for “Multiprotein Complexes”

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PubMed2005

Assay and functional analysis of dynamin-like Mx proteins.

Mx proteins are interferon-induced large guanosine triphosphatases (GTPases) that share structural and functional properties with dynamin and dynamin-like proteins, such as self-assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a range of RNA viruses, including influenza viruses and members of the bunyavirus family. These viruses are inhibited at an early stage in their life cycle, soon after host cell entry and before genome amplification. The association of the human MxA GTPase with membranes of the endoplasmic reticulum seems to support its antiviral function by providing an interaction platform that facilitates viral target recognition, MxA oligomerization, and missorting of the resulting multiprotein complex into large intracellular aggregates.

3T3 Cells↗

PubMed2006

KSR regulation of the Raf-MEK-ERK cascade.

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that functions as a molecular scaffold to enhance signaling between the core kinase components of the ERK cascade--Raf, MEK, and ERK. KSR interacts constitutively with MEK and translocates from the cytosol to the plasma membrane on Ras activation. At the membrane, KSR coordinates the assembly of a multiprotein complex containing Raf, MEK, and ERK and facilitates signal transmission from Raf to MEK and ERK. In this chapter, we will describe methods for assessing KSR function in response to Ras pathway activation. Protocols will be included that examine the ERK scaffolding activity and subcellular localization of KSR.

Animals↗

PubMed2006

Measurement of chromosomal DNA single-strand breaks and replication fork progression rates.

Chromosomal single-strand breaks (SSBs) are the most common lesions arising in cells, but are normally rapidly repaired by multiprotein complexes centered around the scaffold protein, XRCC1. Here, we describe protocols to measure chromosomal SSBs in cells and for recovering and identifying novel components of SSBR complexes in vitro and in vivo. We also describe an assay we employ to measure the rate of replication fork progression in mammalian/vertebrate cells in the presence or absence of DNA damage.

Animals↗

PubMed2006

Analysis of DNA recombination and repair proteins in living cells by photobleaching microscopy.

DNA double strand break repair through homologous recombination has been shown biochemically to require the coordinated action of the RAD52 group of proteins, including the DNA strand exchange protein Rad51. We have started to develop experimental tools to investigate the close cooperation of homologous recombination proteins in living cells, where proteins operate in the context of chromatin and in the presence of other nuclear processes. This chapter describes in detail methods to establish cell lines stably expressing green fluorescent protein -tagged recombination proteins and photobleaching techniques to investigate the behavior of the proteins with the use of live cell video microscopy. Fluorescence recovery after photobleaching (FRAP), fluorescence loss after photobleaching (FLIP), and their combination in the same cell are useful techniques to gain insights into the dynamic behavior of the recombination proteins. Parameters such as diffusion rates and mobile versus immobile fractions before and after DNA damage induction can be obtained. In addition, residence times of recombination proteins at sites of DNA damage can be determined. Through the application of FRAP and FLIP it is possible to establish whether proteins are present in the same multiprotein complex, whether this is affected by DNA damage induction, and whether proteins dynamically associate with and dissociate from sites of DNA damage.

Animals↗

PubMed2003

Herpes simplex virus type-1: a model for genome transactions.

In many respects, HSV-1 is the prototypic herpes virus. However, HSV-1 also serves as an excellent model system to study genome transactions, including DNA replication, homologous recombination, and the interaction of DNA replication enzymes with DNA damage. Like eukaryotic chromosomes, the HSV-1 genome contains multiple origins of replication. Replication of the HSV-1 genome is mediated by the concerted action of several virus-encoded proteins that are thought to assemble into a multiprotein complex. Several host-encoded factors have also been implicated in viral DNA replication. Furthermore, replication of the HSV-1 genome is known to be closely associated with homologous recombination that, like in many cellular organisms, may function in recombinational repair. Finally, recent data have shed some light on the interaction of essential HSV-1 replication proteins, specifically its DNA polymerase and DNA helicases, with damaged DNA.

DNA Damage↗

PubMed1999

PriA: at the crossroads of DNA replication and recombination.

PriA is a single-stranded DNA-dependent ATPase, DNA translocase, and DNA helicase that was discovered originally because of its requirement in vitro for the conversion of bacteriophage phi X174 viral DNA to the duplex replicative form. Studies demonstrated that PriA catalyzes the assembly of a primosome, a multiprotein complex that primes DNA synthesis, on phi X174 DNA. The primosome was shown to be capable of providing both the DNA unwinding function and the Okazaki fragment priming function required for replication fork progression. However, whereas seven proteins, PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG, were required for primosome assembly on phi X174 DNA, only DnaB, DnaC, and DnaG were required for replication from oriC, suggesting that the other proteins were not involved in chromosomal replication. Strains carrying priA null mutations, however, were constitutively induced for the SOS response, and were defective in homologous recombination, repair of UV-damaged DNA, and double-strand breaks, and both induced and constitutive stable DNA replication. The basis for this phenotype can now be explained by the ability of PriA to load replication forks at a D loop, an intermediate that forms during homologous recombination, double-strand break-repair, and stable DNA replication. Thus, a long-theorized connection between recombination and replication is demonstrated.

Base Sequence↗

PubMed1994

Isolation of cytoskeletal proteins from Drosophila.

In this summary of methods, we have attempted to present a series of protocols relevant to the study of proteins that associate with each other inside cells. These techniques have found their main application, in our hands, to cytoskeletal structures, but the utility of these techniques is limited to this application. As we learn more about the physiology and organization of eukaryotic cells, information from a variety of studies and systems indicates that many processes occur in a highly spatially organized manner in cells, using multiprotein complexes. The techniques described here will find application to the study of how organization and associations between proteins contribute to many of cellular processes.

Animals↗

PubMed1996

Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression.

The budding yeast SKP1 gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro. Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes. skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function. skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase. Identification of Skp1p homologs from C. elegans, A. thaliana, and H. sapiens indicates that SKP1 is evolutionarily highly conserved. Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery.

Alleles↗

PubMed1998

Degradation signal masking by heterodimerization of MATalpha2 and MATa1 blocks their mutual destruction by the ubiquitin-proteasome pathway.

Proteolysis by the ubiquitin-proteasome pathway is often regulated, but the mechanisms underlying such regulation remain ill-defined. In Saccharomyces cerevisiae, cell type is controlled by the MAT transcription factors. The alpha2 repressor is a known ubiquitin pathway substrate in alpha haploid cells. We show that a1 is rapidly degraded in a haploids. In a/alpha diploids, alpha2 and a1 are stabilized by heterodimerization. Association depends on N-terminal coiled-coil interactions between a1 and alpha2. Residues in alpha2 important for these interactions overlap a critical determinant of an alpha2 degradation signal, which we delimit by extensive mutagenesis. Our data provide a detailed description of a natural ubiquitin-dependent degradation signal and point to a molecular mechanism for regulated turnover in which proteolytic signals are differentially masked in alternative multiprotein complexes.

Amino Acid Sequence↗

PubMed1998

A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3.

The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.

COP9 Signalosome Complex↗

PubMed2001

XRCC1 stimulates human polynucleotide kinase activity at damaged DNA termini and accelerates DNA single-strand break repair.

XRCC1 protein is required for DNA single-strand break repair and genetic stability but its biochemical role is unknown. Here, we report that XRCC1 interacts with human polynucleotide kinase in addition to its established interactions with DNA polymerase-beta and DNA ligase III. Moreover, these four proteins are coassociated in multiprotein complexes in human cell extract and together they repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. Strikingly, XRCC1 stimulates the DNA kinase and DNA phosphatase activities of polynucleotide kinase at damaged DNA termini and thereby accelerates the overall repair reaction. These data identify a novel pathway for mammalian single-strand break repair and demonstrate a concerted role for XRCC1 and PNK in the initial step of processing damaged DNA ends.

Animals↗

PubMed2003

COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases.

COP9 Signalosome (CSN) is a fascinating protein complex whose biochemical and physiological functions are only beginning to be understood. It is conserved throughout eukaryotes and is critical to the proper development of all multicellular organisms in which its function has been explored. Recent work suggests that CSN plays a key role in sustaining the activity of SCF and other cullin-based ubiquitin ligases, which may account for its essential roles in development. Here, we summarize what is known about CSN, and discuss hypotheses for how CSN promotes the activity of SCF ubiquitin ligases.

Animals↗

PubMed2004

APPL proteins link Rab5 to nuclear signal transduction via an endosomal compartment.

Signals generated in response to extracellular stimuli at the plasma membrane are transmitted through cytoplasmic transduction cascades to the nucleus. We report the identification of a pathway directly linking the small GTPase Rab5, a key regulator of endocytosis, to signal transduction and mitogenesis. This pathway operates via APPL1 and APPL2, two Rab5 effectors, which reside on a subpopulation of endosomes. In response to extracellular stimuli such as EGF and oxidative stress, APPL1 translocates from the membranes to the nucleus where it interacts with the nucleosome remodeling and histone deacetylase multiprotein complex NuRD/MeCP1, an established regulator of chromatin structure and gene expression. Both APPL1 and APPL2 are essential for cell proliferation and their function requires Rab5 binding. Our findings identify an endosomal compartment bearing Rab5 and APPL proteins as an intermediate in signaling between the plasma membrane and the nucleus.

Binding Sites↗

PubMed2000

HSNF5/INI1 gene mutations in lymphoid malignancy.

hSNF5/INI1 is one of the components of the SWI/SNF multiprotein complex that is necessary for the transcriptional activation of several genes and functions by altering chromatin structure. This gene has been thought to be one of the tumor suppressor genes (TSGs) because deletions or mutations were reported in malignant rhabdoid tumors and atypical teratoid and rhabdoid tumors. To evaluate the hSNF5/INI1 gene as a TSG in lymphoid malignancies, we performed a mutational analysis in 23 patients with non-Hodgkin lymphoma (NHL), 24 with acute lymphoblastic leukemia (ALL), 24 with multiple myeloma (MM), 24 with adult T-cell lymphoma/leukemia (ATLL), and 19 with lymphoid cell lines, by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis. Nonsense and missense mutations were found in 1 NHL case and 2 cell lines. Mutations from this NHL case proved to be somatic in origin. These data indicated that alterations in the hSNF5/INI1 gene might be involved in the pathogenesis of lymphoid malignancies.

Base Sequence↗

PubMed2001

LIS1: from cortical malformation to essential protein of cellular dynamics.

The LIS1 gene was cloned following the study of children with lissencephaly and cytogenetic abnormalities involving chromosome 17p, however, the role of the LIS1 protein in normal cortical development is not precisely defined. LIS1 is a component of evolutionarily conserved intracellular multiprotein complexes and recent literature shows that these complexes are essential, not only for neuronal migration, but they might also be fundamental components of the machinery for cell proliferation and intracellular transport.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2000

An in vitro system for the enzymological analysis of avian hepatitis B virus replication and inhibition in core particles.

A detailed analysis of the hepatitis B virus (HBV) replication reaction is important both in understanding viral biology and in developing effective antiviral drugs. This can best be achieved by studying the viral reverse transcriptase (RT) in its natural context, encapsidated within viral core particles in a multiprotein complex, rather than as an isolated enzyme. In order to facilitate a precise enzymological analysis of the avian HBV-RT reaction and its inhibition within replicating cores, a scheme for the purification and analysis of intracellular core particles derived from infected liver tissue has been devised, optimized and evaluated. The purification scheme itself is simple and rapid, and results in preparations with a 25-fold increase in endogenous polymerase activity that persists for over 5 h under assay conditions. In order to assess the suitability of these preparations for mechanistic studies, a thorough evaluation of purity was undertaken, revealing predominantly pure viral protein and nucleic acid, free of contaminating cellular polymerases and phosphatase activities that potently degrade nucleotides and antiviral drugs. Parameters governing optimal polymerase activity have been determined, and an assay for DHBV-RT activity has been developed which offers the highest purity and specific polymerase activity currently available to study hepadnaviral replication and inhibition.

Animals↗

PubMed2001

VAM-1: a new member of the MAGUK family binds to human Veli-1 through a conserved domain.

The MAGUKs (membrane-associated guanylate kinase homologues) constitute a family of peripheral membrane proteins that function in tumor suppression and receptor clustering by forming multiprotein complexes containing distinct sets of transmembrane, cytoskeletal, and cytoplasmic signaling proteins. Here, we report the characterization of the human vam-1 gene that encodes a novel member of the p55 subfamily of MAGUKs. The complete cDNA sequence of VAM-1, tissue distribution of its mRNA, genomic structure, chromosomal localization, and Veli-1 binding properties are presented. The vam-1 gene is composed of 12 exons and spans approx. 115 kb. By fluorescence in situ hybridization the vam-1 gene was localized to 7p15-21, a chromosome region frequently disrupted in some human cancers. VAM-1 mRNA was abundant in human testis, brain, and kidney with lower levels detectable in other tissues. The primary structure of VAM-1, predicted from cDNA sequencing, consists of 540 amino acids including a single PDZ domain near the N-terminus, a central SH3 domain, and a C-terminal GUK (guanylate kinase-like) domain. Sequence alignment, heterologous transfection, GST pull-down experiments, and blot overlay assays revealed a conserved domain in VAM-1 that binds to Veli-1, the human homologue of the LIN-7 adaptor protein in Caenorhabditis. LIN-7 is known to play an essential role in the basolateral localization of the LET-23 tyrosine kinase receptor, by linking the receptor to LIN-2 and LIN-10 proteins. Our results therefore suggest that VAM-1 may function by promoting the assembly of a Veli-1 containing protein complex in neuronal as well as epithelial cells.

Amino Acid Sequence↗

PubMed1998

Cloning and heterogeneous in vivo expression of Tat binding protein-1 (TBP-1) in the mouse.

Tat binding protein-1 (TBP-1) is one of the molecules that interact with HIV Tat protein and have influence on Tat-mediated transactivation of HIV. In addition, TBP-1 has been recognized as a component of the 19S regulatory subunit of the multiprotein complex, the 26S proteasome, that is essential for many basic events in cells, e.g. cell cycle regulation, by degrading the ubiquitinized proteins. Here we have cloned a mouse TBP-1, confirmed its inhibitory action on Tat activity and revealed its in vivo heterogeneous expression pattern in the mouse. The cloned mouse TBP-1 cDNA is 1569 bp in size, longer than reported human TBP-1 cDNA, and another possible initiation site has been identified. Robust expression of TBP-1 mRNA can be observed in the testis, especially in the spermatogonia and spermatocytes. Our immunohistochemical study has revealed that TBP-1 is mainly localized in the nuclei of these testicular cells. Expression of TBP-1 mRNA in CD4+ lymphocytes is confirmed by RT-PCR. Localization of TBP-1 transcripts in vivo is similar to the reported distribution of constitutive components of the 20S proteasome. The heterogeneous and restricted expression of TBP-1 in vivo suggests that there may be a diverse reaction of tissues against the HIV proliferation and a diverse capability of tissues in degrading ubiquitinized proteins in vivo.

ATPases Associated with Diverse Cellular Activitie↗