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The Tec kinases are required for full Ca(2+) mobilization in lymphocytes. Recent data suggest that this process occurs via a multiprotein complex that includes LAT and SLP-76 in T cells and BLNK/SLP-65 in B cells. Mutational analyses have revealed critical roles for Tec kinases in lymphocyte development and function.
The Tec kinases are implicated as important components of the antigen receptor signaling required for proper lymphocyte activation and development. Recent data suggest that these kinases contribute to multiprotein complexes containing LAT and SLP-76 in T cells, and BLNK/SLP-65 in B cells, which are required for activation of PLC-gamma and downstream pathways.
Presenilins are needed for proteolytic processing of transmembrane proteins of the Notch/Lin-12 family and for cleavage of the amyloid precursor protein. Accumulating evidence now strongly implicates Presenilin as the catalytic core of a multiprotein complex that executes an unusual intramembranous cleavage of its substrates. In the case of amyloid precursor protein, this cleavage contributes to the generation of small, toxic amyloid peptides that trigger the pathological development of Alzheimer's disease. In the Notch/Lin-12 pathway, Presenilin-mediated cleavage of the receptor is a crucial feature of ligand-induced receptor activation and signal transduction. In this pathway, the Presenilins perform a regulated cleavage event that follows additional processing steps during receptor maturation and ligand-induced ectodomain removal.
Our understanding of the role of hemidesmosomes in cell-substratum adhesion has greatly improved both as a result of targeted gene mutation experiments and by means of observations of several blistering disorders of the skin in which the absence or defects of hemidesmosomal proteins have been demonstrated. Functionally important domains within the proteins that constitute hemidesmosomes have recently been identified by transfection and mutagenesis studies. These multiprotein complexes appear not only to mediate cell adhesion, but also to transduce signals from the extracellular matrix to the cell interior that may profoundly modulate cell behavior.
The past year has seen significant advances in our understanding of the structure and function of yeast ARS elements. These elements, some of which function as chromosomal origins of DNA replication, are modular in structure. An essential domain, the ARS consensus sequence, binds a multiprotein complex that might be the long-sought initiator protein. The flanking domain contains a DNA unwinding element and a binding site for a multifunctional protein that acts as a replication enhancer.
During the past year, the characterization of mechanisms and factors capable of disrupting nucleosomes during transcriptional activation has been a recurrent theme in studies which address the contribution of nucleosome structure to gene regulation. In vivo studies using yeast and Drosophila together with biochemical purification schemes using nucleosome perturbation assays have provided evidence for the existence of multiprotein complexes that are able to alleviate nucleosome repression. At the same time, new insights into the mechanism of heterochromatin formation have been gained, which have direct links to nucleosome structure.
Genes of the Polycomb and trithorax groups (PcG and trxG) are part of a cellular memory system that maintains inactive and active states of homeotic gene expression in Drosophila. Recent genetic evidence indicates that several related loci in mammals are also involved in the regulation of Hox genes. Like their Drosophila counterparts, the vertebrate gene products are components of multiprotein complexes that regulate transcriptional activation, repression and aspects of chromatin structure. Initial indications suggest the existence of a large mammalian PcG and trxG family, with a potential to encode multiple specialised functions in cell fate and cell-cycle control.
Recent advances highlight two important chromatin remodeling systems involved in the transcriptional process. One system includes several members of the evolutionarily conserved SWI2/SNF2 family found in distinct multiprotein complexes with ATP-dependent nucleosome destabilizing activity; the other is the enzymatic system that governs histone acetylation and deacetylation. Identification of the catalytic subunits of these opposing histone-modifying activities reveal conserved proteins defined genetically as transcriptional regulators.
Major recent advances in hardware performance, sample-handling procedures and software algorithms now allow reliable and sensitive mass spectrometric identification of proteins. Mass spectrometry vastly outperforms traditional sequencing technologies and thereby greatly facilitates the elucidation of the functions of individual proteins as well as multiprotein complexes and larger protein assemblages.
'Cohesin' is a highly conserved multiprotein complex thought to be the primary effector of sister-chromatid cohesion in all eukaryotes. Cohesin complexes in budding yeast hold sister chromatids together from S phase until anaphase, but in metazoans, cohesin proteins dissociate from chromosomes and redistribute into the whole cell volume during prophase, well before sister chromatids separate (reviewed in [1,2]). Here we address this apparent anomaly by investigating the cell-cycle dynamics of DRAD21, the Drosophila orthologue of the Xenopus XRAD21 and Saccharomyces cerevisiae Scc1p/Mcd1p cohesins [3]. Analysis of DRAD21 in S2 Drosophila tissue culture cells and live embryos expressing a DRAD21-green fluorescent protein (GFP) fusion revealed the presence of four distinct subcellular pools of DRAD21: a cytoplasmic pool; a chromosome-associated pool which dissociates from chromatin as chromosomes condense in prophase; a short-lived centrosome-associated pool present during metaphase-anaphase; and a centromere-proximal pool which remains bound to condensed chromosomes, is found along the junction of sister chromatids between kinetochores, and persists until the metaphase-anaphase transition. We conclude that in Drosophila, and possibly all metazoans, a minor pool of cohesin remains bound to centromere-proximal chromatin after prophase and maintains sister-chromatid cohesion until the metaphase-anaphase transition.
BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.
BACKGROUND: Y14 is an RNA binding protein which is part of a multiprotein complex, the exon-exon junction complex (EJC), that assembles on the exon-exon junctions of mRNAs produced by splicing. The position-specific binding of Y14 persists on mRNAs after their export to the cytoplasm. Thus, Y14, together with its interacting proteins, has the capacity to communicate to the cytoplasm the processing history of the mRNA, including the position of the removed introns, information that is likely to be important for defining premature termination codons. How Y14 and other components of the EJC are removed from mRNAs into the cytoplasm has not been determined. RESULTS: We show that Y14 but not another EJC component, Aly/REF, is present in polysome profile fractions containing one ribosome per mRNA. Using reporter constructs in an in vitro splicing/translation-coupled system, we show that Y14 remains associated with untranslated mRNAs but is removed from translationally active mRNAs. Importantly, mRNAs whose translation in vivo is prevented by the presence of strong secondary 5' UTR structure retain Y14 in the cytoplasm. CONCLUSIONS: These findings indicate that Y14 remains associated with mRNAs in the cytoplasm until they are translated, and translation is required to remove Y14 from mRNAs. Thus, the process of translation removes the splicing-dependent EJC protein imprints, which most likely function in the surveillance of mRNAs to define premature termination codons and possibly also in modulating the translation activity of cytoplasmic mRNAs.
gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.
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The ubiquitination and destruction of cyclins provides the way out of mitosis. A multiprotein complex polyubiquitinates cyclin B and promotes the separation of sister chromatids.
BACKGROUND: The orientation of the mitotic spindle influences the asymmetric distribution of cytoplasmic determinants and the positioning of the sibling cell, and therefore has important influences on cell-fate determination and patterning of the embryo. Both the establishment of an axis of polarity and the adjustment of this axis with respect to the coordinates of the embryo have to be controlled. None of the genes identified so far that are involved in these processes seems to have been conserved between flies and nematodes. RESULTS: Here, we show that the bazooka gene encodes a protein with three putative protein-interaction motifs known as PDZ domains and is the first Drosophila representative of the par gene family of Caenorhabditis elegans, members of which are required for establishment of anterior-posterior polarity of the nematode embryo. The bazooka RNA and protein were found to be restricted to the apical cortical cytoplasm of epithelial cells and neuroblasts. Embryos that were mutant for bazooka frequently failed to coordinate the axis of cell polarity with that of the embryo. This was manifested as defective spindle orientation and mispositioning of the daughter cell after division. CONCLUSIONS: The Drosophila gene bazooka is likely to be part of a regulatory mechanism required to coordinate the axis of polarity of a cell with that of the embryo. The PDZ domains of Bazooka provide several protein-protein interfaces, which possibly participate in the assembly of a multiprotein complex at the apical pole.
Important clues to how the mammalian cerebral cortex develops are provided by the analysis of genetic diseases that cause cortical malformations [1-5]. People with Miller-Dieker syndrome (MDS) or isolated lissencephaly sequence (ILS) have a hemizygous deletion or mutation in the LIS1 gene [3,6]; both conditions are characterized by a smooth cerebral surface, a thickened cortex with four abnormal layers, and misplaced neurons [7,8]. LIS1 is highly expressed in the ventricular zone and the cortical plate [9,10], and its product, Lis1, has seven WD repeats [3]; several proteins with such repeats have been shown to interact with other polypeptides, giving rise to multiprotein complexes [11]. Lis1 copurifies with platelet-activating factor acetylhydrolase subunits alpha 1 and alpha 2 [12], and with tubulin; it also reduces microtubule catastrophe events in vitro [13]. We used a yeast two-hybrid screen to isolate new Lis1-interacting proteins and found a mammalian ortholog of NudC, a protein required for nuclear movement in Aspergillus nidulans [14]. The specificity of the mammalian NudC-Lis1 interaction was demonstrated by protein-protein interaction assays in vitro and by co-immunoprecipitation from mouse brain extracts. In addition, the murine mNudC and mLis1 genes are coexpressed in the ventricular zone of the forebrain and in the cortical plate. The interaction of Lis1 with NudC, in conjunction with the MDS and ILS phenotypes, raises the possibility that nuclear movement in the ventricular zone is tied to the specification of neuronal fates and thus to cortical architecture.