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.     

Sequential tethering of Golgins and catalysis of SNAREpin assembly by the vesicle-tethering protein p115

p115 tethers coat protein (COP)I vesicles to Golgi membranes. The acidic COOH-terminal domain of p115 links the Golgins, Giantin on COPI vesicles, to GM130 on Golgi membranes. We now show that a SNARE motif-related domain within p115 stimulates the specific assembly of endogenous Golgi SNAREpins containing the t-SNARE, syntaxin 5. p115 catalyzes the construction of a cognate GOS-28-syntaxin-5 (v-/t-SNARE) complex by first linking the SNAREs to promote their direct interaction. These events are essential for NSF-catalyzed reassembly of postmitotic Golgi vesicles and tubules into mature cisternae. Staging experiments reveal that the linking of Golgins precedes SNAREpin assembly. Thus, p115 coordinates sequential tethering and docking of COPI vesicles by first using long tethers (Golgins) and then short tethers (SNAREs).     

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.     

The membrane-tethering protein p115 interacts with GBF1, an ARF guanine-nucleotide-exchange factor

The membrane-transport factor p115 interacts with diverse components of the membrane-transport machinery. It binds two Golgi matrix proteins, a Rab GTPase, and various members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Here, we describe a novel interaction between p115 and Golgi-specific brefeldin-A-resistant factor 1 (GBF1), a guanine-nucleotide exchange factor for ADP ribosylation factor (ARF). GBF1 was identified in a yeast two-hybrid screen, using full-length p115 as bait. The interaction was confirmed biochemically, using in vitro and in vivo assays. The interacting domains were mapped to the proline-rich region of GBF1 and the head region of p115. These proteins colocalize extensively in the Golgi and in peripheral vesicular tubular clusters. Mutagenesis analysis indicates that the interaction is not required for targeting GBF1 or p115 to membranes. Expression of the p115-binding (pro-rich) region of GBF1 leads to Golgi disruption, indicating that the interaction between p115 and GBF1 is functionally relevant.     

The p115-interactive proteins GM130 and giantin participate in endoplasmic reticulum-Golgi traffic

The transport factor p115 is essential for endoplasmic reticulum (ER) to Golgi traffic. P115 interacts with two Golgi proteins, GM130 and giantin, suggesting that they might also participate in ER-Golgi traffic. Here, we show that peptides containing the GM130 or the giantin p115 binding domain and anti-GM130 and anti-giantin antibodies inhibit transport of vesicular stomatitis virus (VSV)-G protein to a mannosidase II-containing Golgi compartment. To determine whether p115, GM130, and giantin act together or sequentially during transport, we compared kinetics of traffic inhibition. Anti-p115, anti-GM130, and anti-giantin antibodies inhibited transport at temporally distinct steps, with the p115-requiring step before the GM130-requiring stage, and both preceding the giantin-requiring stage. Examination of the distribution of the arrested VSV-G protein showed that anti-p115 antibodies inhibited transport at the level of vesicular-tubular clusters, whereas anti-GM130 and anti-giantin antibodies inhibited after the VSV-G protein moved to the Golgi complex. Our results provide the first evidence that GM130 and giantin are required for the delivery of a cargo protein to the mannosidase II-containing Golgi compartment. These data are most consistent with a model where transport from the ER to the cis/medial-Golgi compartments requires the action of p115, GM130, and giantin in a sequential rather than coordinate mechanism.     

The lck tyrosine protein kinase interacts with the cytoplasmic tail of the CD4 glycoprotein through its unique amino-terminal domain

The CD4 lymphocyte surface glycoprotein and the lck tyrosine protein kinase p56lck are found as a complex in T lymphocytes. We have defined the domains in both proteins that are responsible for this interaction by coexpressing hybrid and deleted forms of the two proteins in HeLa cells. We have found that the unique 32 amino-terminal residues of p56lck and the 38 carboxy-terminal residues of CD4 that comprise the cytoplasmic domain are both necessary and sufficient by themselves for the interaction of the two proteins. The interaction appears to be independent of other T cell-specific proteins and probably occurs before CD4 reaches the cell surface. Our findings suggest that the specialized amino-terminal domains of other members of the src family of intracellular tyrosine kinases may also mediate transmembrane signaling via coupling to the cytoplasmic domains of specific transmembrane proteins.     

Menin interacts directly with the homeobox-containing protein Pem

Scalarane-type sesterterpenes, PHC-1-PHC-7, which have been isolated from a marine sponge, increased hemoglobin production in human chronic myelogenous leukemia cell line K562 at the concentration of 0.1-5 microg/ml. PHC-1, the major constituent, induced the expression of glycophorin A and the enucleation for K562 cells. These sesterterpenes were found to induce erythroid differentiation in K562 cells. In addition, PHC-1 induced G1 arrest of the cell cycle of K562 cells.     

Human cellular prion protein interacts directly with clusterin protein

Prion protein is a glycosyl-phosphatidyl-inositol anchored glycoprotein localized on the surface and within a variety of cells. Its conformation change is thought to be essential for the proliferation of prion neurodegenerative diseases. Using the yeast two-hybrid assay we identified an interaction between prion protein and clusterin, a chaperone glycoprotein. This interaction was confirmed in a mammalian system by in vivo co-immunoprecipitation and in vitro by circular dichroism analysis. Through deletion mapping analysis we demonstrated that the alpha subunit, but not the beta subunit, of clusterin binds to prion and that the C-terminal 62 amino acid segment of the putative alpha helix region of clusterin is essential for the binding interaction. The full prion protein as well as the N-terminal section (aa 23-95) and C-terminal (aa 96-231) were shown to interact with clusterin. These findings provide new insights into the molecular mechanisms of interaction between prion and clusterin protein and contribute to the understanding of prion protein's physiological function.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.     

The early divisome protein FtsA interacts directly through its 1c subdomain with the cytoplasmic domain of the late divisome protein FtsN

In Escherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsN(Cyto)), a single transmembrane domain (FtsN(TM)), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directly in vitro. FtsN(Cyto) is sufficient to bind to FtsA, but only when it is tethered to FtsN(TM) or to a leucine zipper. Mutation of a conserved patch of positive charges in FtsN(Cyto) to negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsN(Cyto-TM) is not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsN(Cyto) protein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.     

The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus

The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood. There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p). The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins. We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins. A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p. Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro. However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus. Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.     

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.     

Gamma-adaptin interacts directly with Rabaptin-5 through its ear domain

In yeast two-hybrid screening using gamma1-adaptin, a subunit of the AP-1 adaptor complex of clathrin-coated vesicles derived from the trans-Golgi network (TGN), as bait, we found that it could interact with Rabaptin-5, an effector of Rab5 and Rab4 that regulates membrane docking with endosomes. Further two-hybrid analysis revealed that the interaction occurs between the ear domain of gamma1-adaptin and the COOH-terminal coiled-coil region of Rabaptin-5. Pull down assay with a fusion protein between glutathione S-transferase and the ear domain of gamma1-adaptin and coimmunoprecipitation analysis revealed that the interaction occurs in vitro and in vivo. Immunocytochemical analysis showed that gamma1-adaptin and Rabaptin-5 colocalize to a significant extent on perinuclear structures, probably on recycling endosomes, and are redistributed into the cytoplasm upon treatment with brefeldin A. These results suggest that the gamma1-adaptin-Rabaptin-5 interaction may play a role in membrane trafficking between the TGN and endosomes.     

The EspB protein of enterohaemorrhagic Escherichia coli interacts directly with alpha-catenin

Enterohaemorrhagic Escherichia coli (EHEC) belongs to a family of pathogens that cause attaching and effacing (A/E) lesion on target cells. The EspB protein of EHEC is translocated both to the host cell cytoplasm and to the membrane, and is essential for the signalling events leading to A/E lesion. To determine the actual role of EspB in this process, we tried to identify the EspB binding partner of the host cell protein, using a yeast two-hybrid assay, and obtained a cytoskeletal-associated protein, α -catenin. The α -catenin bound directly to the N-terminal region of EspB, both in solid (overlay assay) and solution (pull-down assay) phases, and it was recruited to the EHEC adherence site, dependent on EspB. Expression of the N-terminal region of EspB, as well as the whole EspB in host cells, inhibited F-actin accumulation on the adherence site. We conclude that EspB recruits α -catenin at the EHEC adherence site by direct interaction, and that the recruitment of α -catenin is essential for EHEC-induced A/E lesion formation.     

The yeast nucleoporin Nup53p specifically interacts with Nic96p and is directly involved in nuclear protein import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of approximately 30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen with NIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.     

Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin

We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y. (2001) J. Biol. Chem. 276, 6867-6873). In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin. The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary. Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex. GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin. Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi. Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.     

The unique amino-terminal domain of p56lck regulates interactions with tyrosine protein phosphatases in T lymphocytes.

The catalytic activity of p56lck is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue (tyrosine 505). Accumulating data show that this phosphorylation is mediated by the tyrosine protein kinase p50csk and that it is reversed by the transmembrane tyrosine protein phosphatase CD45. Recent studies have indicated that dephosphorylation of tyrosine 505 in resting T cells is necessary for the initiation of antigen-induced T-cell activation. To better understand this phenomenon, we have characterized the factors regulating tyrosine 505 phosphorylation in an antigen-specific T-cell line (BI-141). As is the case for other T-cell lines, Lck molecules from unstimulated BI-141 cells exhibited a pronounced dephosphorylation of the inhibitory carboxyl-terminal tyrosine. This state could be corrected by incubation of cells with the tyrosine protein phosphatase inhibitor pervanadate, suggesting that it reflected the unrestricted action of tyrosine protein phosphatases. In structure-function analyses, mutation of the site of Lck myristylation (glycine 2) partially restored phosphorylation at tyrosine 505 in BI-141 cells. Since the myristylation-defective mutant also failed to stably associate with cellular membranes, this effect was most probably the consequence of removal of p56lck from the vicinity of membrane phosphatases like CD45. Deletion of the unique domain of Lck, or its replacement by the equivalent sequence from p59fyn, also increased the extent of tyrosine 505 phosphorylation in vivo. This effect was unrelated to changes in Lck membrane association and therefore was potentially related to defects in crucial protein-protein interactions at the membrane. In contrast, deletion of the SH3 or SH2 domain, or mutation of the phosphotransfer motif (lysine 273) or the site of autophosphorylation (tyrosine 394), had no impact on phosphate occupancy at tyrosine 505. In combination, these results indicated that the hypophosphorylation of the inhibitory tyrosine of p56(lck) in T lymphocytes is likely the result of the predominant action of tyrosine protein phosphatases. Moreover, they showed that both the amino-terminal myristylation signal and the unique domain of p56(lck) play critical roles in this process.