Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER.

The role of rab33b, a Golgi-specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST-rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin-5 and rabex-5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin-5 and rabex-5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.     

Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.     

Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and λ repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.     

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.     

Identification and characterization of Iporin as a novel interaction partner for rab1

Background  

The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport.     

Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner

Reduced expression of the 56-kDa human selenium binding protein-1 (hSP56) has been reported in many types of human malignancies, including prostate, lung, ovarian, thyroid and colorectal cancers. hSP56 also has been implicated in selenium-dependent cell growth inhibition. However, the molecular basis of hSP56’s function has not been elucidated. In the present study, we identified von Hippel-Lindau protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein partner of hSP56 using a yeast two-hybrid screen. The interaction between hSP56 and VDU1 was confirmed by yeast two-hybrid analysis and in vitro binding experiments. hSP56 and VDU1 co-localized in the perinuclear region of LNCaP human prostate cancer cells. The full-length VDU1 specifically interacted with a selenium-replete form of hSP56. We also demonstrate stable incorporation of selenium into hSP56, in a mode distinct from conventional selenocysteine-containing selenoproteins. These findings suggest that hSP56 may play a role in ubiquitination/deubiquitination-mediated protein degradation pathways in a selenium-dependent manner.     

BIM1 Encodes a Microtubule-binding Protein in Yeast

A previously uncharacterized yeast gene (YER016w) that we have named BIM1 (binding to microtubules) was obtained from a two-hybrid screen of a yeast cDNA library using as bait the entire coding sequence of TUB1 (encoding α-tubulin). Deletion of BIM1 results in a strong bilateral karyogamy defect, hypersensitivity to benomyl, and aberrant spindle behavior, all phenotypes associated with mutations affecting microtubules in yeast, and inviability at extreme temperatures (i.e., ≥37°C or ≤14°C). Overexpression of BIM1 in wild-type cells is lethal. A fusion of Bim1p with green fluorescent protein that complements the bim1Δ phenotypes allows visualization in vivo of both intranuclear spindles and extranuclear microtubules in otherwise wild-type cells. A bim1 deletion displays synthetic lethality with deletion alleles of bik1, num1, and bub3 as well as a limited subset of tub1 conditional-lethal alleles. A systematic study of 51 tub1 alleles suggests a correlation between specific failure to interact with Bim1p in the two-hybrid assay and synthetic lethality with the bim1Δ allele. The sequence of BIM1 shows substantial similarity to sequences from organisms across the evolutionary spectrum. One of the human homologues, EB1, has been reported previously as binding APC, itself a microtubule-binding protein and the product of a gene implicated in the etiology of human colon cancer.     

A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.     

Identification of the COMM-domain containing protein 1 as specific binding partner for the guanine-rich RNA sequence binding factor 1

BackgroundThe guanine-rich RNA sequence binding factor 1 (GRSF1) is an RNA-binding protein of the hnRNP H/F family, which has been implicated in erythropoiesis, regulation of the redox homeostasis, embryonic brain development, mitochondrial function and cellular senescence. The molecular basis for GRSF1-RNA interaction has extensively been studied in the past but for the time being GRSF1 binding proteins have not been identified.MethodsTo search for GRSF1 binding proteins we first employed the yeast two-hybrid system and screened a cDNA library of human fetal brain for potential GRSF1 binding proteins. Subsequently, we explored the protein-protein-interaction of the recombiant proteins, carried out immunoprecipitation experiments to confirm the interaction of the native proteins in living cells and performed truncation studies to identify the protein-binding motif of GRSF1.ResultsUsing the yeast two-hybrid system we identified the COMM-domain containing protein 1 (COMMD1) as specific GRSF1 binding protein and in vitro truncation studies suggested that COMMD1 interacts with the alanine-rich domain of GRSF1. Co-immunoprecipitation strategies indicated that COMMD1-GRSF1 interaction was RNA independent and also occurred in living cells expressing the two native proteins.ConclusionIn mammalian cells the COMM-domain containing protein 1 (COMMD1) specifically interacts with the Ala-rich domain of GRSF1 in an RNA-independent manner.General significanceThis is the first report describing a specific GRSF1 binding protein.     

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure

Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.

Structured summary of protein interactions

GM130 and Piltcolocalize by fluorescence microscopy (View interaction)Arf6(Q67L)physically interacts with Pilt by two hybrid (View Interaction: 1, 2)Piltphysically interacts with Arf6(Q67L) by pull down (View interaction)     

The Membrane Transport Factor TAP/p115 Cycles between the Golgi and Earlier Secretory Compartments and Contains Distinct Domains Required for Its Localization and Function

The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al., 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15°C treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi.TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH₂-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH₂-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.     

The Nucleolar PICT-1/GLTSCR2 Protein Forms Homo-Oligomers

The human “protein interacting with carboxyl terminus 1” (PICT-1), also designated as the “glioma tumor suppressor candidate region 2 gene product”, GLTSCR2, is a nucleolar protein whose activity is, as yet, unknown. Contradictory results regarding the role of PICT-1 in cancer have been reported, and PICT-1 has been suggested to function either as a tumor suppressor protein or as an oncogene. In this study, we demonstrate self-association of PICT-1. Through yeast two-hybrid assay, we identified PICT-1 as its own interaction partner. We confirmed the interaction of PICT-1 with itself by direct yeast two-hybrid assay and also showed self-association of PICT-1 in mammalian cells by co-immunoprecipitation and fluorescence resonance energy transfer assays. Furthermore, we confirmed direct self-association of PICT-1 by using in vitro microfluidic affinity binding assays. The later assay also identified the carboxy-terminal domain as mediating self-interaction of PICT-1. Glutaraldehyde cross-linking and gel-filtration assays suggest that PICT-1 forms dimers, though it may form higher-order complexes as well. Our findings add another layer of complexity in understanding the different functions of PICT-1 and may help provide insights regarding the activities of this protein.     

Na/K-ATPase beta1-subunit associates with neuronal growth regulator 1 (NEGR1) to participate in intercellular interactions

Neuronal growth regulator 1 (NEGR1) is a GPI-anchored membrane protein that is involved in neural cell adhesion and communication. Multiple genome wide association studies have found that NEGR1 is a generic risk factor for multiple human diseases, including obesity, autism, and depression. Recently, we reported that Negr1^−/− mice showed a highly increased fat mass and affective behavior. In the present study, we identified Na/K-ATPase, beta1-subunit (ATP1B1) as an NEGR1 binding partner by yeast two-hybrid screening. NEGR1 and ATP1B1 were found to form a relatively stable complex in cells, at least partially co-localizing in membrane lipid rafts. We found that NEGR1 binds with ATP1B1 at its C-terminus, away from the binding site for the alpha subunit, and may contribute to intercellular interactions. Collectively, we report ATP1B1 as a novel NEGR1-interacting protein, which may help deciphering molecular networks underlying NEGR1-associated human diseases.     

Molecular Cloning and Characterization of Rab27a and Rab27b,Novel Human Rab Proteins Shared by Melanocytes and Platelets

Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes. Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.     

A unique residue in rab3c determines the interaction with novel binding protein Zwint-1

Exocytic events are tightly regulated cellular processes in which rab GTPases and their interacting proteins perform an important function. We set out to identify new binding partners of rab3, which mediates regulated secretion events in specialized cells. We discovered Zwint-1 as a rab3 specific binding protein that bound preferentially to rab3c. The interaction depends on a critical residue in rab3c that determines the binding efficiency of Zwint-1, which is immaterial for interaction with rabphilin3a. Rab3c and Zwint-1 are expressed highly in brain and colocalized extensively in primary hippocampal neurons. We also found that SNAP25 bound to the same region in Zwint-1 as rab3c, suggesting a new role for the kinetochore protein Zwint-1 in presynaptic events that are regulated by rab3 and SNAP25.     

Mapping the interaction between GRASP65 and GM130, components of a protein complex involved in the stacking of Golgi cisternae.

The nature of the complex containing GRASP65, a membrane protein involved in establishing the stacked structure of the Golgi apparatus, and GM130, a putative Golgi matrix protein and vesicle docking receptor, was investigated. Gel filtration revealed that GRASP65 and GM130 interact in detergent extracts of Golgi membranes under both interphase and mitotic conditions, and that this complex can bind to the vesicle docking protein p115. Using in vitro translation and site-directed mutagenesis in conjunction with immunoprecipitation, the binding site for GRASP65 on GM130 was mapped to the sequence xxNDxxxIMVI-COOH at the C-terminus of GM130, a region known to be required for its localization to the Golgi apparatus. The same approach was used to show that the binding site for GM130 on GRASP65 maps to amino acids 189-201, a region conserved in the mammalian and yeast proteins and reminiscent of PDZ domains. Using green fluorescent protein (GFP)-tagged reporter constructs, it was shown that one essential function of the interaction between GRASP65 and GM130 is in the correct targeting of the two proteins to the Golgi apparatus.     

Combining Natural Sequence Variation with High Throughput Mutational Data to Reveal Protein Interaction Sites

Many protein interactions are conserved among organisms despite changes in the amino acid sequences that comprise their contact sites, a property that has been used to infer the location of these sites from protein homology. In an inter-species complementation experiment, a sequence present in a homologue is substituted into a protein and tested for its ability to support function. Therefore, substitutions that inhibit function can identify interaction sites that changed over evolution. However, most of the sequence differences within a protein family remain unexplored because of the small-scale nature of these complementation approaches. Here we use existing high throughput mutational data on the in vivo function of the RRM2 domain of the Saccharomyces cerevisiae poly(A)-binding protein, Pab1, to analyze its sites of interaction. Of 197 single amino acid differences in 52 Pab1 homologues, 17 reduce the function of Pab1 when substituted into the yeast protein. The majority of these deleterious mutations interfere with the binding of the RRM2 domain to eIF4G1 and eIF4G2, isoforms of a translation initiation factor. A large-scale mutational analysis of the RRM2 domain in a two-hybrid assay for eIF4G1 binding supports these findings and identifies peripheral residues that make a smaller contribution to eIF4G1 binding. Three single amino acid substitutions in yeast Pab1 corresponding to residues from the human orthologue are deleterious and eliminate binding to the yeast eIF4G isoforms. We create a triple mutant that carries these substitutions and other humanizing substitutions that collectively support a switch in binding specificity of RRM2 from the yeast eIF4G1 to its human orthologue. Finally, we map other deleterious substitutions in Pab1 to inter-domain (RRM2–RRM1) or protein-RNA (RRM2–poly(A)) interaction sites. Thus, the combined approach of large-scale mutational data and evolutionary conservation can be used to characterize interaction sites at single amino acid resolution.     

Molecular cloning of two novel rab genes from human melanocytes

We isolated the genes of two small GTP-binding proteins of the rab family from a human melanocyte cDNA library and from melanoma cells. One gene, rab30 codes for a novel rab protein of 203 amino acids with minimal homology to previously documented GTPases. The other, rab22b, appears to be an isoform of the human homologue of canine rab22. Both rab mRNAs displayed a nearly ubiquitous pattern of expression in the various tissues examined. Rab22b and rab30 were mapped to chromosomes 18 and 11, respectively.