LRCH Proteins: A Novel Family of Cytoskeletal Regulators

Background

Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs.

Principal Findings

Taking advantage of the existence of a single LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures.

Conclusion/Significance

These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations.     

A Novel Family of Ubiquitous Heavy Metal Ion Transport Proteins

We describe a novel diverse family of metal ion transporter (CDF) proteins (the cation diffusion facilitator (CDF) family) with members occurring in both prokaryotes and eukaryotes. Thirteen sequenced protein members of the CDF family have been identified, several of which have been shown to transport cobalt, cadmium and/or zinc. All members of the CDF family possess six putative transmembrane spanners with strongest conservation in the four N-terminal spanners, and on the basis of the analyses, we present a unified structural model. Members of the family are shown to exhibit an unusual degree of size variation, sequence divergence, and differences in cell localization and polarity. The phylogenetic tree for the CDF family reveals that prokaryotic and eukaryotic proteins cluster separately. It allows functional predictions for some uncharacterized members of this family. A signature sequence specific for the CDF family is derived. Received: 15 July 1996/Revised: 21 October 1996     

A Novel Bipartite Centrosome Coordinates the Apicomplexan Cell Cycle

Apicomplexan parasites can change fundamental features of cell division during their life cycles, suspending cytokinesis when needed and changing proliferative scale in different hosts and tissues. The structural and molecular basis for this remarkable cell cycle flexibility is not fully understood, although the centrosome serves a key role in determining when and how much replication will occur. Here we describe the discovery of multiple replicating core complexes with distinct protein composition and function in the centrosome of Toxoplasma gondii. An outer core complex distal from the nucleus contains the TgCentrin1/TgSfi1 protein pair, along with the cartwheel protein TgSas-6 and a novel Aurora-related kinase, while an inner core closely aligned with the unique spindle pole (centrocone) holds distant orthologs of the CEP250/C-Nap protein family. This outer/inner spatial relationship of centrosome cores is maintained throughout the cell cycle. When in metaphase, the duplicated cores align to opposite sides of the kinetochores in a linear array. As parasites transition into S phase, the cores sequentially duplicate, outer core first and inner core second, ensuring that each daughter parasite inherits one copy of each type of centrosome core. A key serine/threonine kinase distantly related to the MAPK family is localized to the centrosome, where it restricts core duplication to once per cycle and ensures the proper formation of new daughter parasites. Genetic analysis of the outer core in a temperature-sensitive mutant demonstrated this core functions primarily in cytokinesis. An inhibition of ts-TgSfi1 function at high temperature caused the loss of outer cores and a severe block to budding, while at the same time the inner core amplified along with the unique spindle pole, indicating the inner core and spindle pole are independent and co-regulated. The discovery of a novel bipartite organization in the parasite centrosome that segregates the functions of karyokinesis and cytokinesis provides an explanation for how cell cycle flexibility is achieved in apicomplexan life cycles.     

PAQR Proteins: A Novel Membrane Receptor Family Defined by an Ancient7-Transmembrane Pass Motif

An emerging series of papers has identified new receptor proteins that predict seven-transmembrane pass topologies. We have consolidated this family to 11 human genes and have named the family PAQR, after two of the initially described ligands (progestin and adipoQ receptors). This protein family has ancient evolutionary roots, with identified homologs found in eubacteria. To date, published data indicate that the prokaryotic members of this family appear to encode hemolysin-type proteins, while in eukaryotes, PAQR proteins encode functional receptors with a broad range of apparent ligand specificities. We provide the complete human and mouse complement of this family, suggest a conserved structure/topology with invariant intracellular amino acid residues, and have measured mRNA expression levels for these genes across a range of human tissues.[Reviewing Editor: Martin Kreitman]     

The Sec1 Family: A Novel Family of Proteins Involved in Synaptic Transmission and General Secretion

Abstract: The Sec1 family, a novel family of proteins involved in synaptic transmission and general secretion, is described. To date, 14 members of this family have been identified: four yeast proteins, Sec1, Sly1, Slp1/Vps33, and Vps45/Stt10; three nematode proteins, Unc-18 and the homologues of Sly1 and Slp1; the Drosophila Rop; and six mammalian proteins, the rat Munc-18/n-Sec1/rbSec1A and rbSec1B, the mouse Munc-18b/muSec1 and Munc-18c, and the bovine Munc-18 and mSec1. The mammalian proteins share 44–63% sequence identity with the nematode Unc-18 and Drosophila Rop proteins and 20–29% with the yeast proteins and their nematode homologues. The Sec1 proteins are mostly hydrophilic and lack a transmembrane domain. Nevertheless, Sec1 proteins are found as membrane-bound proteins. Some of them are also found as soluble, cytoplasmic proteins. Binding of the rat brain Sec1 to the presynaptic membrane may be due to strong interaction with syntaxin, an integral component of this membrane. The rat brain Sec1 is also bound to Cdk5, a neural cyclin-dependent kinase. The Sec1 proteins play a positive role in exocytosis. Loss of function mutations in SEC1 , SLY1 , or SLP1 result in blocking of protein transport between distinct yeast subcellular compartments. Inactivation of unc-18 and rop results in inhibition of neurotransmitter release and, in the case of rop , inhibition of general secretion as well. In addition, studies of Rop and n-Sec1 indicate that they also play a negative role in synaptic transmission, mediated by their interaction with syntaxin. A working model addressing the dual regulative role of the Sec1 proteins in secretion is presented.     

The Inner Membrane Complex Sub-compartment Proteins Critical for Replication of the Apicomplexan Parasite Toxoplasma gondii Adopt a Pleckstrin Homology Fold

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.     

Bcl-2家族蛋白及其在细胞凋亡中的作用

Bcl-2家族蛋白是目前已知的细胞凋亡中最重要的调控因子,在细胞凋亡通路中起着重要的调节作用．对其作用机制的研究将有助于对肿瘤,自身免疫性和神经变性等疾病的治疗。该文介绍Bcl-2家族中主要的几种蛋白在凋亡中的作用,以及对线粒体膜通透性的调控作用。     

A Novel Role for VICKZ Proteins in Maintaining Epithelial Integrity during Embryogenesis

Background

VICKZ (IGF2BP1,2,3/ZBP1/Vg1RBP/IMP1,2,3) proteins bind RNA and help regulate many RNA-mediated processes. In the midbrain region of early chick embryos, VICKZ is expressed in the neural folds and along the basal surface of the neural epithelium, but, upon neural tube closure, is down-regulated in prospective cranial neural crest (CNC) cells, concomitant with their emigration and epithelial-to-mesenchymal transition (EMT). Electroporation of constructs that modulate cVICKZ expression demonstrates that this down-regulation is both necessary and sufficient for CNC EMT. These results suggest that VICKZ down-regulation in CNC cell-autonomously promotes EMT and migration. Reduction of VICKZ throughout the embryo, however, inhibits CNC migration non-cell-autonomously, as judged by transplantation experiments in Xenopus embryos.

Results and Conclusions

Given the positive role reported for VICKZ proteins in promoting cell migration of chick embryo fibroblasts and many types of cancer cells, we have begun to look for specific mRNAs that could mediate context-specific differences. We report here that the laminin receptor, integrin alpha 6, is down-regulated in the dorsal neural tube when CNC cells emigrate, this process is mediated by cVICKZ, and integrin alpha 6 mRNA is found in VICKZ ribonucleoprotein complexes. Significantly, prolonged inhibition of cVICKZ in either the neural tube or the nascent dermomyotome sheet, which also dynamically expresses cVICKZ, induces disruption of these epithelia. These data point to a previously unreported role for VICKZ in maintaining epithelial integrity.     

植物GSKs:新发现的具有多种重要功能的基因家族

糖原合成酶激酶 (GSK 3)是一种高度保守的丝氨酸 苏氨酸蛋白激酶 ,在动物中参与诸如糖原合成、胰岛素调节、多种蛋白的转录激活和发育调控等许多生命活动的信号转导。在植物中也分离到了GSK 3 Like基因 ,在拟南芥中的GSKs家族分为四种。GSKs家族在植物中也扮演着重要的角色 ,现有的证据表明 ,植物GSKs可能参与植物的渗透胁迫应答、伤害应答以及油菜素内酯信号转导 ,调节花的发育等等一系列生命活动进程。讨论植物GSKs的发现及其功能研究的最新进展。     

A Family of Novel DNA-Binding Nuclear Proteins Having Polypyrimidine Tract-Binding Motif and Arginine/Serine-Rich Motif

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220α and NP220β) have all functional domains mentioned above while two shorter forms (NP220γ and NP220δ) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3′-splicing site of specific genes in response to differentiation-dependent signals.     

The Physiological Properties of a Novel Family of VDAC-Like Proteins from Drosophila melanogaster

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (β-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.     

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication.     

A Novel Class of RanGTP Binding Proteins

The importin-α/β complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of ∼20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-β. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-β, it prevents the activation of Ran''s GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-β, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.The nuclear pore complexes (NPC)¹ are the sites where the exchange of macromolecules between nucleus and cytoplasm occurs (Feldherr et al., 1984). Transport through the NPCs is bidirectional and comprises a multitude of substrates. Small molecules can passively diffuse through the NPC. The transport of proteins and RNAs >40–60 kD is, however, generally an active process, i.e., it is energy dependent (Newmeyer et al., 1986) and mediated by saturable transport receptors (Goldfarb et al., 1986; Michaud and Goldfarb, 1991; Jarmolowski et al., 1994). To accomplish multiple rounds of transport, these transport receptors are thought to shuttle between nucleus and cytoplasm (Goldfarb et al., 1986). An import receptor, for example, has to bind its import substrate initially in the cytoplasm, release it after NPC passage into the nucleus, and return to the cytoplasm without the cargo. Conversely, an export factor has to bind the export substrate only in the nucleus and on the way out. This model predicts asymmetry in these transport cycles and implies that the binding of the transport receptor to its cargo is regulated by the different environments of nucleus and cytoplasm.The nuclear import of proteins with a classical nuclear localization signal (NLS) is to date the best characterized nucleocytoplasmic transport pathway (for reviews see Görlich and Mattaj, 1996; Koepp and Silver, 1996; Schlenstedt, 1996). The signal contains one or more clusters of basic amino acids (for review see Dingwall and Laskey, 1991) and is recognized by the importin-α/β complex (for references see Sweet and Gerace, 1995; Panté and Aebi, 1996). The α subunit provides the NLS binding site (Imamoto et al., 1995; Weis et al., 1995) and is therefore also called the NLS receptor (Adam and Gerace, 1991). The β subunit accounts for the interaction with the NPC (Görlich et al., 1995; Moroianu et al., 1995) and carries importin-α with the NLS substrate into the nucleus. The translocation into the nucleus is terminated by the disassembly of the importin complex, and both subunits are returned probably separately to the cytoplasm. Importin-α interacts with -β via its importin-β binding domain (IBB domain; Görlich et al., 1996a ; Weis et al., 1996a ). Binding to importin-β with an IBB domain is sufficient for nuclear entry, and the IBB domain can therefore be regarded as the nuclear targeting signal of importin-α. The export domain of importin-α has not yet been identified, but it is distinct from the IBB domain.The small GTPase Ran (Drivas et al., 1990; Bischoff and Ponstingl, 1991b ; Belhumeur et al., 1993) plays a key role in NLS-dependent protein import (Melchior et al., 1993; Moore and Blobel, 1993). GTP hydrolysis by Ran is absolutely essential for import (Melchior et al., 1993; Moore and Blobel, 1993; Schlenstedt et al., 1995a ; Palacios et al., 1996) and is possibly even its sole source of energy (Weis et al., 1996b ). Although the molecular mechanism of import is far from being understood, it appears that Ran fulfils at least two distinct functions in this process: first, Ran''s GTP cycle probably drives translocation into the nucleus (Melchior et al., 1993; Moore and Blobel, 1993; Weis et al., 1996b ), which appears to involve the binding of (cytoplasmic) RanGDP to the NPC, followed by nucleotide exchange and GTP hydrolysis, but it does not involve binding of RanGTP to importin-β (Görlich et al., 1996b ). Unfortunately, nothing is known of how Ran''s GTP cycle would translate into a directed movement through the NPC. Secondly, Ran regulates the interaction between importin-α and -β (Rexach and Blobel, 1995; Chi et al., 1996; Görlich et al., 1996b ). Binding of RanGTP to importin-β disassembles the importin-α/β complex at the nuclear side of the NPC, thereby terminating translocation (Görlich et al., 1996b ). The asymmetric distribution of Ran''s principal GDP/GTP exchange factor (RCC1; Bischoff and Ponstingl, 1991a ) and GTPase activating protein (RanGAP1, or RNA1 in yeast; Bischoff et al., 1995a ; Becker et al., 1995) crucially determines where the importin heterodimer can form and where it is forced to dissociate. RCC1 is a nuclear, chromatin-bound protein (Ohtsubo et al., 1987, 1989) that generates RanGTP in the nucleus, whereas free RanGTP is depleted from the cytoplasm by RanGAP1, which is excluded from the nucleoplasm (Hopper et al., 1990; Matunis et al., 1996; Mahajan et. al, 1997). Thus, low RanGTP levels in the cytoplasm allow importin-α to bind -β, and the high RanGTP concentration in the nuclear compartment dissociates the importin complex. The concentration of free RanGTP can, in this model, be regarded as a marker for cytoplasmic identity (low RanGTP) and nuclear identity (high RanGTP), which is “sensed” by the Ran-binding site in importin-β.It is likely that at least some properties of importin-β are shared by the mediators of the other nucleocytoplasmic transport pathways. This is emphasized by the recent identification of the importin-β–related transportin (Pollard et al., 1996) as an import receptor recognizing the M9 domain, the nuclear targeting signal in hnRNP A1 (Michael et al., 1995), and of yeast transportin (Kap 104p) as an import receptor for mRNA binding proteins (Aitchison et al., 1996). Furthermore, importin-β or its NPC-binding domain cross-compete with other pathways, such as M9-dependent import, NES-mediated nuclear export, and the export of mRNA and U snRNA (Kutay et al., 1997). This would suggest that these other transport receptors share at least some binding sites at the NPC and take a similar path through the nuclear pore complex as importin-β.In addition to importin-β, a number of other Ran-binding proteins are detectable in eukaryotic cells, e.g., in overlay blots using Ran γ-[³²P]GTP as a probe. These can be grouped into two classes (Lounsbury et al., 1994, 1996): first, those with a RanBP1 homology domain including the Ran binding protein 1 (RanBP1) itself (Coutavas et al., 1993; Bischoff et al., 1995b ) and the nuclear pore protein RanBP2, which has four RanBP1 homology domains (Wu et al., 1995; Yokoyama et al., 1995). Their binding to Ran can be competed by RanBP1. Second, importin-β and so far unidentified protein(s) of ∼120 kD whose Ran-binding is competed by importin-β but not by excess of RanBP1 (Lounsbury et al., 1994, 1996). Both RanBP1 and importin-β inhibit the nucleotide exchange on RanGTP (Coutavas et al., 1993; Lounsbury et al., 1994, 1996; Bischoff et al., 1995b ; Görlich et al., 1996b ). However, they do not cross-compete with each other for Ran binding but instead bind to different, nonoverlapping sites on Ran (Chi et al., 1996; Kutay et al., 1997; Lounsbury and Macara, 1997). Another striking difference is that RanBP1 facilitates the activation of Ran''s GTPase by RanGAP1 (Beddow et al., 1995; Bischoff et al., 1995b ), whereas the importin-β/RanGTP complex is entirely GAP resistant (Floer and Blobel, 1996; Görlich et al., 1996b ).Although a direct involvement of Ran has so far only been demonstrated in the importin-dependent transport pathway, perturbations in the Ran system have severe effects on a great variety of cellular functions, such as RNA processing, RNA export, regulation of chromosome structure, cell cycle progression, initiation of replication, microtubule structure, etc. (for review see Dasso, 1993; Sazer, 1996). One could argue that these effects are all secondary consequences from an impaired NLS-dependent protein import. However, it is also possible that these defects are more direct and that eukaryotic cells contain many immediate targets of Ran function.Here we describe a novel superfamily of Ran-binding proteins, which includes about a dozen factors in yeast and probably even more in higher eukaryotes. The members of this superfamily share with importin-β an NH₂-terminal sequence motif that appears to account for RanGTP binding. Indeed we could confirm the interaction with Ran for the following factors: RanBP7 and RanBP8, two novel, related proteins described here, Cse1p, a cell cycle regulator in yeast, CAS, which is required for apoptosis in cultured human cells, and for Msn5p and Pse1p from yeast. Of these we have characterized RanBP7 and RanBP8 in more detail. Both resemble closely importin-β in their interaction with Ran, and both bind directly to nuclear pore complexes. RanBP7 can cross the nuclear membrane rapidly and in both directions. We provide evidence for a transport cycle in which RanBP7 first enters the nucleus, binds RanGTP inside the nucleus as a prerequisite for rapid re-export to the cytoplasm, after which the RanBP7/RanGTP complex becomes finally disassembled by the concerted action of RanBP1 and RanGAP1 in the cytoplasm. We propose that during these transport cycles, RanBP7 would normally carry an as yet unidentified cargo. This means, RanBP7 and possibly also the other members of the RanBP7/Cse1p/ importin-β superfamily could function as transport receptors that shuttle between nucleus and cytoplasm. RanBP7 and importin-β form an abundant, heterodimeric complex in the cytoplasm that appears to have a function different from nuclear import of proteins with a classical NLS. It might be a way to regulate either RanBP7 or importin-β function. Alternatively, the RanBP7/importin-β complex might constitute an import receptor with a substrate specificity different from that of the importin-α/β complex.