Association of Arabidopsis type-II ROPs with the plasma membrane requires a conserved C-terminal sequence motif and a proximal polybasic domain

Plant ROPs (or RACs) are soluble Ras-related small GTPases that are attached to cell membranes by virtue of the post-translational lipid modifications of prenylation and S-acylation. ROPs (RACs) are subdivided into two major subgroups called type-I and type-II. Whereas type-I ROPs terminate with a conserved CaaL box and undergo prenylation, type-II ROPs undergo S-acylation on two or three C-terminal cysteines. In the present work we determined the sequence requirement for association of Arabidopsis type-II ROPs with the plasma membrane. We identified a conserved sequence motif, designated the GC-CG box, in which the modified cysteines are flanked by glycines. The GC-CG box cysteines are separated by five to six mostly non-polar residues. Deletion of this sequence or the introduction of mutations that change its nature disrupted the association of ROPs with the membrane. Mutations that changed the GC-CG box glycines to alanines also interfered with membrane association. Deletion of a polybasic domain proximal to the GC-CG box disrupted the plasma membrane association of AtROP10. A green fluorescent protein fusion protein containing the C-terminal 25 residues of AtROP10, including its polybasic domain and GC-CG box, was primarily associated with the plasma membrane but a similar fusion protein lacking the polybasic domain was exclusively localized in the soluble fraction. These data provide evidence for the minimal sequence required for plasma membrane association of type-II ROPs in Arabidopsis and other plant species.     

Efficient T-cell receptor signaling requires a high-affinity interaction between the Gads C-SH3 domain and the SLP-76 RxxK motif

The relationship between the binding affinity and specificity of modular interaction domains is potentially important in determining biological signaling responses. In signaling from the T-cell receptor (TCR), the Gads C-terminal SH3 domain binds a core RxxK sequence motif in the SLP-76 scaffold. We show that residues surrounding this motif are largely optimized for binding the Gads C-SH3 domain resulting in a high-affinity interaction (K(D)=8-20 nM) that is essential for efficient TCR signaling in Jurkat T cells, since Gads-mediated signaling declines with decreasing affinity. Furthermore, the SLP-76 RxxK motif has evolved a very high specificity for the Gads C-SH3 domain. However, TCR signaling in Jurkat cells is tolerant of potential SLP-76 crossreactivity, provided that very high-affinity binding to the Gads C-SH3 domain is maintained. These data provide a quantitative argument that the affinity of the Gads C-SH3 domain for SLP-76 is physiologically important and suggest that the integrity of TCR signaling in vivo is sustained both by strong selection of SLP-76 for the Gads C-SH3 domain and by a capacity to buffer intrinsic crossreactivity.     

Plant-specific mitotic targeting of RanGAP requires a functional WPP domain

The small GTPase Ran is involved in nucleocytoplasmic transport, spindle formation, nuclear envelope (NE) formation, and cell-cycle control. In vertebrates, these functions are controlled by a three-dimensional gradient of Ran-GTP to Ran-GDP, established by the spatial separation of Ran GTPase-activating protein (RanGAP) and the Ran guanine nucleotide exchange factor RCC1. While this spatial separation is established by the NE during interphase, it is orchestrated during mitosis by association of RCC1 with the chromosomes and RanGAP with the spindle and kinetochores. SUMOylation of vertebrate RanGAP1 is required for NE, spindle, and centromere association. Arabidopsis RanGAP1 (AtRanGAP1) lacks the SUMOylated C-terminal domain of vertebrate RanGAP, but contains a plant-specific N-terminal domain (WPP domain), which is necessary and sufficient for its targeting to the NE in interphase. Here we show that the human and plant RanGAP-targeting domains are kingdom specific. AtRanGAP1 has a mitotic trafficking pattern uniquely different from that of vertebrate RanGAP, which includes targeting to the outward-growing rim of the cell plate. The WPP domain is necessary and sufficient for this targeting. Point mutations in conserved residues of the WPP domain also abolish targeting to the nuclear rim and the cell plate, suggesting that the same mechanism is involved in both targeting events. These results indicate that plant and animal RanGAPs undergo different migration patterns during cell division, which require their kingdom-specific targeting domains.     

Upstream polybasic region in peptides enhances dual specificity for prenylation by both farnesyltransferase and geranylgeranyltransferase type I

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of a farnesyl or geranylgeranyl lipid, respectively, near the C-terminus of their protein substrates. FTase and GGTase I differ in both their substrate specificity and magnesium dependence, where the activity of FTase, but not GGTase I, is activated by magnesium. Many protein substrates of these enzymes contain an upstream polybasic region that is proposed to increase the affinity of the substrate and aid in plasma membrane association. Here, we demonstrate that the addition of an upstream polybasic region to a peptide substrate enhances the binding affinity of FTase approximately 4-fold for the peptide but diminishes the catalytic efficiency of the reaction, reflected by decreases in both the prenylation rate constant and kcat/KM. Specifically, the prenylation rate constant decreases 7-fold at 5 mM MgCl2 for the peptide KKKSKTKCVIM (C-terminal sequence of K-Ras4B) in comparison to TKCVIM. This decrease is accompanied by an alteration in the dependence on magnesium, as the K(Mg) increases from 2.2 +/- 0.1 mM for TKCVIM to 11.5 +/- 0.1 mM for KKKSKTKCVIM. The presence of an upstream polybasic region does not significantly affect GGTase I-catalyzed reactions, as only minimal changes are seen in Kd, kcat/KM, and k(chem) values. Thus, the presence of an upstream polybasic region enhances the dual prenylation of these substrates, by decreasing the catalytic efficiency of farnesylation catalyzed by FTase to a level comparable to that of geranylgeranylation catalyzed by GGTase I.     

Control of photoreceptor cell fate by the sevenless protein requires a functional tyrosine kinase domain

The sevenless (sev) gene determines the fate of a single photoreceptor cell type in the eye of Drosophila. It encodes a putative cell-surface protein with homology to tyrosine kinases. Here we have determined the complete structure of the sev gene and have demonstrated that the role of the sev protein in this developmental decision is critically dependent on the tyrosine kinase function. In comparison with other known tyrosine kinases, the sev gene product is unique in size and structure. It is a polypeptide of 2554 amino acids with two putative transmembrane segments. A single amino acid substitution in the ATP-binding site of the putative kinase domain results in the synthesis of an inactive sev protein unable to determine cell fate.     

Stable nucleotide binding to DnaA requires a specific glutamic acid residue within the AAA+ box II motif

In complex with ATP, but not ADP, DnaA protein multimers unwind a specific region of duplex DNA within the chromosomal replication origin, oriC, triggering a series of reactions that result in initiation of DNA replication. Following replication initiation, ATP hydrolysis, which is coupled to DNA replication, results in the generation of initiation-incompetent ADP-DnaA. Suppression of overinitiation of replication requires that ADP-DnaA complexes be stably maintained until the next round of replication. Thus, the functional and structural requirements that ensure stable nucleotide binding to DnaA are crucial for proper regulation of replication. Here, we demonstrate that Glu143 of DnaA, located within the AAA+ box II N-linker motif, is a key residue involved in stable nucleotide binding. A Glu143 substitution variant of DnaA (DnaA E143A) bound to ADP on ice with an affinity similar to wild-type DnaA, but the resultant ADP-DnaA E143A complex was more labile at 37 °C than wild-type ADP-DnaA complexes. Consistent with this, conversion of ADP-DnaA E143A to ATP-DnaA E143A was stimulated at 37°C in the presence of ATP, which also stimulated replication of a minichromosome in an in vitro reconstitution reaction. Expression of DnaA E143A in vivo inhibited cell growth in an oriC-dependent manner, suggesting that DnaA E143A caused over-initiation of replication, consistent with the in vitro results. Glu is a highly conserved residue at the corresponding position of γ-proteobacterial DnaA orthologs. Our finding of the novel role for the DnaA N-linker region may represent a conserved function of this motif among those DnaA orthologs.     

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7,8-dihydro-8-oxo-2′-deoxyguanosine:2′-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1–Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.     

植物一个新box H/ACA snoRNA的鉴定及功能分析

通过生物信息学方法对拟南芥基因组序列进行搜索,发现两个新的非编码小分子RNA基因,分别命名为AthsnoR206 a和AthsnoR206b。它们相距约170nt,位于蛋白质基因间隔区。MFOLD二级结构预测这两个RNA均具有典型的box H/ACAsnoRNA"发夹-铰链-发夹-尾巴"结构,符合box H/ACA snoRNA的判定标准;两个RNA分子的反义序列一致,可以判定它们为同一基因的两个拷贝。分析预测snoR206的两段反义序列分别指导拟南芥rRNA小亚基U1717位点和大亚基U2181位点的假尿嘧啶化修饰。在其它13种包括单子叶植物和双子叶植物在内的植物搜索到14个snoR206同源分子,其中12个发现于表达序列标签中,表明该snoRNA在植物中表达且广泛存在。具有双功能的snoR206在人和酵母中的部分功能同源分子分别为U70和snR32,表明其祖先分子在进化过程中存在分子重组。     

The C2 domain calcium-binding motif: structural and functional diversity.

The C2 domain is a Ca(2+)-binding motif of approximately 130 residues in length originally identified in the Ca(2+)-dependent isoforms of protein kinase C. Single and multiple copies of C2 domains have been identified in a growing number of eukaryotic signalling proteins that interact with cellular membranes and mediate a broad array of critical intracellular processes, including membrane trafficking, the generation of lipid-second messengers, activation of GTPases, and the control of protein phosphorylation. As a group, C2 domains display the remarkable property of binding a variety of different ligands and substrates, including Ca2+, phospholipids, inositol polyphosphates, and intracellular proteins. Expanding this functional diversity is the fact that not all proteins containing C2 domains are regulated by Ca2+, suggesting that some C2 domains may play a purely structural role or may have lost the ability to bind Ca2+. The present review summarizes the information currently available regarding the structure and function of the C2 domain and provides a novel sequence alignment of 65 C2 domain primary structures. This alignment predicts that C2 domains form two distinct topological folds, illustrated by the recent crystal structures of C2 domains from synaptotagmin 1 and phosphoinositide-specific phospholipase C-delta 1, respectively. The alignment highlights residues that may be critical to the C2 domain fold or required for Ca2+ binding and regulation.     

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae

The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. Here we conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/Gse and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in class C Vps complex components. We find that Tor1 does not regulate the known function of the class C Vps complex in protein sorting. Instead class C vps mutants fail to recover from rapamycin-induced growth arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more effective than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact class C Vps complex is required to mediate intracellular amino acid homeostasis for efficient Tor signaling.     

Transformation by Fos proteins requires a C-terminal transactivation domain.

The Fos family of proteins now includes seven members: the retroviral proteins FBR-v-Fos and FBJ-v-Fos and the cellular proteins c-Fos, FosB, FosB2, Fra1, and Fra2. Four proteins (FBR-v-Fos, FBJ-v-Fos, c-Fos, and FosB) transform established rodent fibroblast cell lines, while three (FosB2, Fra1, and Fra2) do not. As all family members display sequence-specific DNA-binding activity as part of a heterodimeric complex with Jun proteins, other features must account for the differences in transforming potential. We demonstrate here that all transforming members have a C-terminal transactivation domain that is lacking in nontransforming members. The nontransforming proteins Fra1 and Fra2 can be converted to transforming proteins by fusion of a transactivation domain from either FosB or VP16. We also demonstrate that differences in the basic region-leucine zipper domain affecting either the affinity or sequence specificity of DNA binding are not determinants of the difference in transforming potential among members of the Fos family. The results further define the functional requirements for transformation by Fos proteins and suggest that the subunit composition of AP1 complexes is an important determinant of mitogenic signalling capability.     

A human protein specific for the immunoglobulin octamer DNA motif contains a functional homeobox domain

The homeobox domain is shared by Drosophila homeotic proteins, yeast mating type proteins, and some functionally uncharacterized mammalian proteins. A lymphoid-restricted human protein that binds to the immunoglobulin octamer regulatory motif was shown to contain an amino acid sequence that has 33% amino acid identity with the consensus sequence of the previously cloned homebox domains. This homeobox gene was localized to chromosome 19, thus mapping separately from other human homebox genes. A mutant protein containing amino acid substitutions within a putative helix-turn-helix motif in the homeobox domain did not bind DNA detectably. This human homeobox protein was shown to bind the same DNA sequence as the homeobox domains of the yeast mating type proteins and Drosophila homeotic protein, suggesting that homeobox proteins may have closely related DNA binding characteristics.     

Assembly of Snu114 into U5 snRNP requires Prp8 and a functional GTPase domain

Snu114 is a U5 snRNP protein essential for pre-mRNA splicing. Based on its homology with the ribosomal translocase EF-G, it is thought that GTP hydrolysis by Snu114 induces conformational rearrangements in the spliceosome. We recently identified allele-specific genetic interactions between SNU114 and genes encoding three other U5 snRNP components, Prp8 and two RNA-dependent ATPases, Prp28 and Brr2, required for destabilization of U1 and U4 snRNPs prior to catalysis. To shed more light onto the function of Snu114, we have now directly analyzed snRNP and spliceosome assembly in SNU114 mutant extracts. The Snu114-60 C-terminal truncation mutant, which is synthetically lethal with the ATPase mutants prp28-1 and brr2-1, assembles spliceosomes but subsequently blocks U4 snRNP release. Conversely, mutants in the GTPase domain fail to assemble U5 snRNPs. These mutations prevent the interaction of Snu114 with Prp8 as well as with U5 snRNA. Since Prp8 is thought to regulate the activity of the DEAD-box ATPases, this strategy of snRNP assembly could ensure that Prp8 activity is itself regulated by a GTP-dependent mechanism.