p115 Rho GTPase activating protein interacts with MEKK1

Mammalian MAP/ERK kinase kinase 1 (MEKK1) was identified as a mammalian homolog of Ste11p of the yeast pheromone-induced mating pathway. Like Ste11p, MEKK1 is a MAP3 kinase linked to at least two MAP kinase cascades and regulatory events that require cytoskeletal reorganization. MEKK1 is activated by molecules that impact cytoskeletal function. MEKK1-/-cells are defective in cell migration, demonstrating that it is required for cell motility. MEKK1 has a 1,200 residue N-terminal regulatory domain that interacts with a dozen identified proteins. Using part of the MEKK1 N-terminus in a yeast two-hybrid screen, we discovered a novel interaction with p115 Rho GTPase-activating protein (GAP). The p115 Rho GAP binds to MEKK1 in vitro and in intact cells. The p115 Rho GAP has selectivity for RhoA over other Rho family members. Expression of p115 Rho GAP reduces MEKK1-induced signaling to AP-1. The reduced activation of AP-1 is dependent on the association of MEKK1 with p115 Rho GAP, because deletion of the Rho GAP SH3 domain, which abrogates their interaction, restores the stimulatory effect of MEKK1 on AP-1 activity. Here we have identified an MEKK1 binding partner that offers a connection between this protein kinase and the machinery regulating cytoskeletal reorganization.     

Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association

Assembly of the cytosolic coat protein I (COPI) complex at the ER-Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t(1/2) 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.     

The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH(2)-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.     

Rpg1p/Tif32p, a subunit of translation initiation factor 3, interacts with actin-associated protein Sla2p

The yeast two-hybrid system was used to screen for proteins that interact in vivo with Saccharomyces cerevisiae Rpg1p/Tif32p, the large subunit of the translation initiation factor 3 core complex (eIF3). Eight positive clones encoding portions of the SLA2/END4/MOP2 gene were isolated. They overlapped in the region of amino acids 318-550. Subsequent deletion analysis of Sla2p showed that amino acids 318-373 were essential for the two-hybrid protein-protein interaction. The N-terminal part of Rpg1p (aa 1-615) was essential and sufficient for the Rpg1p-Sla2p interaction. A coimmunoprecipitation assay provided additional evidence for the physical interaction of Rpg1p/Tif32p with Sla2p in vivo. Using immunofluorescence microscopy, Rpg1p and Sla2p proteins were colocalized at the patch associated with the tip of emerging bud. Considering the essential role of Rpg1p as the large subunit of the eIF3 core complex and the association of Sla2p with the actin cytoskeleton, a putative role of the Rpg1p-Sla2p interaction in localized translation is discussed.     

Human proliferation-related protein p120 interacts with HSRP1

Protein p120 is a proliferation-related nucleolar protein, which is detectable early in the G1-phase of the cell cycle and peaks early in the S-phase. Most human malignant tumor cells contain much higher levels of protein p120 than do normal resting cells. To learn more about p120-associated proteins, a yeast two-hybrid screen was carried out using protein p120 as the bait. Five positive clones were identified from 2 million clones for further analysis. Three of them encoded portions of the same protein, which had identity to human SRP1. The recombinant p120 and HSRP1 proteins produced in Sf9 cells are assocated with each other, confirming the results of the yeast genetic assay. Protein SRP1 was originally characterized as a suppressor of RNA polymerase I mutations, and recently human SPR1 was identified as a receptor for nuclear localization sequences. In the present study, use of deletion mutations revealed that the binding of human SRP1 to p120 required the p120 nuclear localization signal (amino acids 96–119) and the C-terminus of human SPR1 (amino acids 453–491). Edited by: S. A. Gerbi     

p14ARF interacts with DAXX: Effects on HDM2 and p53

The p14ARF (ARF) tumour suppressor plays an important role in the cellular response to oncogene activation. In this report, we demonstrate an interaction between ARF and DAXX, a highly conserved protein with identified roles in the regulation of gene expression. HDM2 was shown to interact with each of ARF and DAXX upon upregulation of expression as well as at lower expression levels following transfection of ARF and DAXX. Through immunofluorescence analysis, we observed that endogenous ARF and DAXX colocalize both to nucleoli and to nuclear bodies in cell lines that co-express both proteins. Similar results were obtained upon co-transfection of ARF and DAXX. Co-expression of ARF and DAXX was further found to inhibit ARF-mediated HDM2 sumoylation and to induce sumoylation and ubiquitination of DAXX itself, implicating DAXX as a substrate of ARF-mediated post-translational events. We also observed induction of p53 sumoylation in the presence of ARF and DAXX, an effect that was inhibited by upregulation of HDM2 expression. In summary, we have identified DAXX as a novel ARF binding partner and substrate of ARF-mediated sumoylation and suggest that DAXX acts as a modifier of both p53-dependent and p53-independent ARF function.     

Depletion of vesicle-tethering factor p115 causes mini-stacked Golgi fragments with delayed protein transport

Depletion of p115 with small interfering RNA caused fragmentation of the Golgi apparatus, resulting in dispersed distribution of stacked short cisternae and a vesicular structure (mini-stacked Golgi). The mini-stacked Golgi with cis- and trans-organization is functional in protein transport and glycosylation, although secretion is considerably retarded in p115 knockdown cells. The fragmented Golgi was further disrupted by treatment with breferdin A and reassembled into the mini-stacked Golgi by removal of the drug, as observed in control cells. In addition, p115 knockdown cells maintained retrograde transport from the Golgi to the endoplasmic reticulum, although the rate was not as efficient as in control cells. While no alternation of microtubule networks was found in p115 knockdown cells, the fragmented Golgi resembled those in cells treated with anti-microtubule drugs. The results suggest that p115 is involved in vesicular transport between endoplasmic reticulum and the Golgi, along with microtubule networks.     

The nuclear export signal of splicing factor Uap56p interacts with nuclear pore-associated protein Rae1p for mRNA export in Schizosaccharomyces pombe

Mammalian UAP56 or its homolog Sub2p in Saccharomyces cerevisiae are members of the ATP-dependent RNA helicase family and are required for splicing and nuclear export of mRNA. Previously we showed that in Schizosaccharomyces pombe Uap56p is critical for mRNA export. It links the mRNA adapter Mlo3p, a homolog of Yra1p in S. cerevisiae or Aly in mammals, to nuclear pore-associated mRNA export factor Rae1p. In this study we show that, in contrast to S. cerevisiae, Uap56p in S. pombe is not required for pre-mRNA splicing. The putative RNA helicase function of Uap56p is not required for mRNA export. However, the RNA-binding motif of Uap56p is critical for nuclear export of mRNA. Within Uap56p we identified nuclear import and export signals that may allow it to shuttle between the nucleus and the cytoplasm. We found that Uap56p interacts with Rae1p directly via its nuclear export signal, and this interaction is critical for the nuclear export activity of Uap56p as well as for exporting mRNA. RNA binding and the ability to shuttle between the nucleus and cytoplasm are important features of mRNA export carriers such as HIV-Rev. Our results suggest that Uap56p could function similarly as an export carrier of mRNA in S. pombe.     

The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.     

p80 coilin,a coiled body-specific protein,interacts with ataxin-1, the SCA1 gene product

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 is associated with an elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system and co-immunoprecipitation experiments, we have found that p80 coilin, coiled body-specific protein, binds to ataxin-1. In further experiments with deletion mutants, we found that the C-terminal regions of ataxin-1 and p80 coilin were essential for this interaction. In HeLa cells that have been co-transfected with ataxin-1 and p80 coilin, the p80 coilin protein co-localizes with ataxin-1 aggregates in the nucleoplasm. However, immunohistochemical analysis and immunofluorescence assays showed that mutant ataxin-1 aggregates do not redistribute p80 coilin's dot-like structures in the Purkinje cells of SCA1 transgenic mice. This feature of the interaction between ataxin-1 and p80 coilin suggests that p80 coilin might be implicated in altering the function of ataxin-1.     

RACK1, a protein kinase C scaffolding protein, interacts with the PH domain of p120GAP

The Ras GTPase-activating protein p120GAP is a multidomain protein consisting of a variety of noncatalytic domains that may be involved in its regulation. RACK1 is a membrane-associated protein that binds the C2 domain of PKC and is related in sequence to the beta subunit of heterotrimeric G-proteins which has been implicated in binding to PH domains. Because p120GAP contains both PH and C2/CaLB domains we determined whether it is also a RACK1 binding protein. Coimmunoprecipitation experiments indicate that p120GAP associates with RACK1, whereas PH or C2/CaLB domain deletion mutants do not. A fusion protein containing the GAP PH domain bound to endogenous RACK1 in lysates in a concentration-dependent manner and directly associated with recombinant RACK1. Finally, serine/threonine phosphorylation appears to be involved in regulating this association. These results suggest that p120GAP and RACK1 interact in vivo in a manner dependent upon both the PH and C2/CaLB domains of GAP.     

The Saccharomyces cerevisiae protein Ccz1p interacts with components of the endosomal fusion machinery

The yeast protein Ccz1p is necessary for vacuolar protein trafficking and biogenesis. In a complex with Mon1p, it mediates fusion of transport intermediates with the vacuole membrane by activating the small GTPase Ypt7p. Additionally, genetic data suggest a role of Ccz1p in earlier transport steps, in the Golgi. In a search for further proteins interacting with Ccz1p, we identified the endosomal soluble N -ethylmaleimide-sensitive factor attachment protein receptor Pep12p as an interaction partner of Ccz1p. Combining the ccz1 Δ mutation with deletions of PEP12 or other genes encoding components of the endosomal fusion machinery, VPS21, VPS9 or VPS45 , results in synthetic growth phenotypes. The genes MON1 and YPT7 also interact genetically with PEP12 . These results suggest that the Ccz1p–Mon1p–Ypt7p complex is involved in fusion of transport vesicles to multiple target membranes in yeast cells.     

The RNA export factor Gle1p is located on the cytoplasmic fibrils of the NPC and physically interacts with the FG-nucleoporin Rip1p, the DEAD-box protein Rat8p/Dbp5p and a new protein Ymr 255p

Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets. In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein.     

Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth

Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.     

The nuclear actin-related protein of Saccharomyces cerevisiae, Arp4, directly interacts with the histone acetyltransferase Esa1p

Ten actin-related proteins are known in Saccharomyces cerevisiae, classified into Arps1-10 according to their relatedness to actin. Arp4, a nuclear protein, essential for viability of S. cerevisiae, is a component of at least three chromatin-modifying complexes, one of which is the histone acetyltransferase (HAT) complex NuA4. Since recent data point to a role for Arp4 in the recruitment to specific sites of interaction, we tested if Arp4 directly interacts with the HAT Esa1p that is the catalytic subunit of NuA4. We observed that Arp4 directly binds to Esa1p, whereas Act1p, which is also a component of the NuA4 complex, does not interact with Esa1p. The interaction of Arp4 and Esa1p was not abolished by a deletion of one or both of the specific insertions present in the ARP4 gene. We propose that the interaction of Arp4 with Esa1p is crucial for proper functioning and targeting of the NuA4 complex.