High-Affinity Binding Of The AP-1 Adaptor Complex to Trans-Golgi Network Membranes Devoid Of Mannose 6-Phosphate Receptors

The GTP-binding protein ADP-ribosylation factor (ARF) initiates clathrin-coat assembly at the trans-Goli network (TGN) by generating high-affinity membrane-binding sites for the AP-1 adaptor complex. Both transmembrane proteins, which are sorted into the assembling coated bud, and novel docking proteins have been suggested to be partners with GTP-bound ARF in generating the AP-1-docking sites. The best characterized, and probably the major transmembrane molecules sorted into the clathrin-coated vesicles that form on the TGN, are the mannose 6-phosphate receptors (MPRs). Here, we have examined the role of the MPRs in the AP-1 recruitment process by comparing fibroblasts derived from embryos of either normal or MPR-negative animals. Despite major alterations to the lysosome compartment in the MPR-deficient cells, the steady-state distribution of AP-1 at the TGN is comparable to that of normal cells. Golgi-enriched membranes prepared from the receptor-negative cells also display an apparently normal capacity to recruit AP-1 in vitro in the presence of ARF and either GTP or GTPγS. The AP-1 adaptor is recruited specifically onto the TGN and not onto the numerous abnormal membrane elements that accumulate within the MPR-negative fibroblasts. AP-1 bound to TGN membranes from either normal or MPR-negative fibroblasts is fully resistant to chemical extraction with 1 M Tris-HCl, pH 7, indicating that the adaptor binds to both membrane types with high affinity. The only difference we do note between the Golgi prepared from the MPR-deficient cells and the normal cells is that AP-1 recruited onto the receptor-lacking membranes in the presence of ARF1·GTP is consistently more resistant to extraction with Tris. Because sensitivity to Tris extraction correlates well with nucleotide hydrolysis, this finding might suggest a possible link between MPR sorting and ARF GAP regulation. We conclude that the MPRs are not essential determinants in the initial steps of AP-1 binding to the TGN but, instead, they may play a regulatory role in clathrin-coated vesicle formation by affecting ARF·GTP hydrolysis.

GCS1, an Arf Guanosine Triphosphatase-activating Protein in Saccharomyces cerevisiae, Is Required for Normal Actin Cytoskeletal Organization In Vivo and Stimulates Actin Polymerization In Vitro

Recent cloning of a rat brain phosphatidylinositol 3,4,5-trisphosphate binding protein, centaurin α, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin α is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin α, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Δ) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Δ was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

Sla1p Is a Functionally Modular Component of the Yeast Cortical Actin Cytoskeleton Required for Correct Localization of Both Rho1p-GTPase and Sla2p, a Protein with Talin Homology

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme β-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.  Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

The PDZ Domain of the LIM Protein Enigma Binds to β-Tropomyosin

PDZ and LIM domains are modular protein interaction motifs present in proteins with diverse functions. Enigma is representative of a family of proteins composed of a series of conserved PDZ and LIM domains. The LIM domains of Enigma and its most related family member, Enigma homology protein, bind to protein kinases, whereas the PDZ domains of Enigma and family member actin-associated LIM protein bind to actin filaments. Enigma localizes to actin filaments in fibroblasts via its PDZ domain, and actin-associated LIM protein binds to and colocalizes with the actin-binding protein α-actinin-2 at Z lines in skeletal muscle. We show that Enigma is present at the Z line in skeletal muscle and that the PDZ domain of Enigma binds to a skeletal muscle target, the actin-binding protein tropomyosin (skeletal β-TM). The interaction between Enigma and skeletal β-TM was specific for the PDZ domain of Enigma, was abolished by mutations in the PDZ domain, and required the PDZ-binding consensus sequence (Thr-Ser-Leu) at the extreme carboxyl terminus of skeletal β-TM. Enigma interacted with isoforms of tropomyosin expressed in C2C12 myotubes and formed an immunoprecipitable complex with skeletal β-TM in transfected cells. The association of Enigma with skeletal β-TM suggests a role for Enigma as an adapter protein that directs LIM-binding proteins to actin filaments of muscle cells.

Isolated Mammalian and Schizosaccharomyces pombe Ran-binding Domains Rescue S. pombe sbp1 (RanBP1) Genomic Mutants

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein–mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1− yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1− yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.

Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal NeuronsV⃞

Dendritic mRNA transport and local translation at individual potentiated synapses may represent an elegant way to form synaptic memory. Recently, we characterized Staufen, a double-stranded RNA-binding protein, in rat hippocampal neurons and showed its presence in large RNA-containing granules, which colocalize with microtubules in dendrites. In this paper, we transiently transfect hippocampal neurons with human Staufen-green fluorescent protein (GFP) and find fluorescent granules in the somatodendritic domain of these cells. Human Stau-GFP granules show the same cellular distribution and size and also contain RNA, as already shown for the endogenous Stau particles. In time-lapse videomicroscopy, we show the bidirectional movement of these Staufen-GFP–labeled granules from the cell body into dendrites and vice versa. The average speed of these particles was 6.4 μm/min with a maximum velocity of 24.3 μm/min. Moreover, we demonstrate that the observed assembly into granules and their subsequent dendritic movement is microtubule dependent. Taken together, we have characterized a novel, nonvesicular, microtubule-dependent transport pathway involving RNA-containing granules with Staufen as a core component. This is the first demonstration in living neurons of movement of an essential protein constituent of the mRNA transport machinery.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni2+-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-Å resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

The ATM homologue MEC1 is required for phosphorylation of replication protein A in yeast

Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein, required for cellular DNA replication, repair, and recombination. In human cells, RPA is phosphorylated during the S and G2 phases of the cell cycle and also in response to ionizing or ultraviolet radiation. Saccharomyces cerevisiae exhibits a similar pattern of cell cycle-regulated RPA phosphorylation, and our studies indicate that the radiation-induced reactions occur in yeast as well. We have examined yeast RPA phosphorylation during the normal cell cycle and in response to environmental insult, and have demonstrated that the checkpoint gene MEC1 is required for the reaction under all conditions tested. Through examination of several checkpoint mutants, we have placed RPA phosphorylation in a novel pathway of the DNA damage response. MEC1 is similar in sequence to human ATM, the gene mutated in patients with ataxia-telangiectasia (A-T). A-T cells are deficient in multiple checkpoint pathways and are hypersensitive to killing by ionizing radiation. Because A-T cells exhibit a delay in ionizing radiation-induced RPA phosphorylation, our results indicate a functional similarity between MEC1 and ATM, and suggest that RPA phosphorylation is involved in a conserved eukaryotic DNA damage-response pathway defective in A-T.

The Golgi-associated COPI-coated buds and vesicles contain β/γ-actin

It has been shown previously that the morphology and subcellular positioning of the Golgi complex is controlled by actin microfilaments. To further characterize the association between actin microfilaments and the Golgi complex, we have used the Clostridium botulinum toxins C2 and C3, which specifically inhibit actin polymerization and cause depolymerization of F-actin in intact cells by the ADP ribosylation of G-actin monomers and the Rho small GTP-binding protein, respectively. Normal rat kidney cells treated with C2 showed that disruption of the actin and the collapse of the Golgi complex occurred concomitantly. However, when cells were treated with C3, the actin disassembly was observed without any change in the organization of the Golgi complex. The absence of the involvement of Rho was further confirmed by the treatment with lysophosphatidic acid or microinjection with the constitutively activated form of RhoA, both of which induced the stress fiber formation without affecting the Golgi complex. Immunogold electron microscopy in normal rat kidney cells revealed that β- and γ-actin isoforms were found in Golgi-associated COPI-coated buds and vesicles. Taken together, the results suggest that the Rho signaling pathway does not directly regulate Golgi-associated actin microfilaments, and that β- and γ-actins might be involved in the formation and/or transport of Golgi-derived vesicular or tubular intermediates.

A novel precursor recognition element facilitates posttranslational binding to the signal recognition particle in chloroplasts

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP/LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts.

Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca2+ act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT−/−) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.

Immunophilins, Refsum disease, and lupus nephritis: The peroxisomal enzyme phytanoyl-COA α-hydroxylase is a new FKBP-associated protein

FKBP52 (FKBP59, FKBP4) is a “macro” immunophilin that, although sharing high structural and functional homologies in its amino-terminal domain with FKBP12 (FKBP1), does not have immunosuppressant activity when complexed with FK506, unlike FKBP12. To investigate the physiological function of FKBP52, we used the yeast two-hybrid system as an approach to find its potential protein partners and, from that, its cellular role. This methodology, which already has allowed us to find the FK506-binding protein (FKBP)-associated protein FAP48, also led to the detection of another FKBP-associated protein. Determination of the sequence of this protein permitted its identification as phytanoyl-CoA α-hydroxylase (PAHX), a peroxisomal enzyme that so far was unknown as an FKBP-associated protein. Inactivation of this enzyme is responsible for Refsum disease in humans. The protein also corresponds to the mouse protein LN1, which could be involved in the progress of lupus nephritis. We show here that PAHX has the physical capacity to interact with the FKBP12-like domain of FKBP52, but not with FKBP12, suggesting that it is a particular and specific target of FKBP52. Whereas the binding of calcineurin to FKBP12 is potentiated by FK506, the specific association of PAHX and FKBP52 is maintained in the presence of FK506. This observation suggests that PAHX is a serious candidate for studying the cellular signaling pathway(s) involving FKBP52 in the presence of immunosuppressant drugs.