bluuebunny/crossref_metadata_2025 huggingface dataset:
Composition and Dynamics of Human Mitochondrial Nucleoids
Authors: Garrido Nuria , Griparic Lorena , Jokitalo Eija , Wartiovaara Jorma , van der Bliek Alexander M. , Spelbrink Johannes N.
Published: April 2003

The organization of multiple mitochondrial DNA (mtDNA) molecules in discrete protein-DNA complexes called nucleoids is well studied inSaccharomyces cerevisiae. Similar structures have recently been observed in human cells by the colocalization of a Twinkle-GFP fusion protein with mtDNA. However, nucleoids in mammalian cells are poorly characterized and are often thought of as relatively simple structures, despite the yeast paradigm. In this article we have used immunocytochemistry and biochemical isolation procedures to characterize the composition of human mitochondrial nucleoids. The results show that both the mitochondrial transcription factor TFAM and mitochondrial single-stranded DNA-binding protein colocalize with Twinkle in intramitochondrial foci defined as nucleoids by the specific incorporation of bromodeoxyuridine. Furthermore, mtDNA polymerase POLG and various other as yet unidentified proteins copurify with mtDNA nucleoids using a biochemical isolation procedure, as does TFAM. The results demonstrated that mtDNA in mammalian cells is organized in discrete protein-rich structures within the mitochondrial network. In vivo time-lapse imaging of nucleoids show they are dynamic structures able to divide and redistribute in the mitochondrial network and suggest that nucleoids are the mitochondrial units of inheritance. Nucleoids did not colocalize with dynamin-related protein 1, Drp1, a protein of the mitochondrial fission machinery.
ADP-Ribosylation Factor 6 and a Functional PIX/p95-APP1 Complex Are Required for Rac1B-mediated Neurite Outgrowth
Authors: Albertinazzi Chiara , Za Lorena , Paris Simona , de Curtis Ivan
Published: April 2003

The mechanisms coordinating adhesion, actin organization, and membrane traffic during growth cone migration are poorly understood. Neuritogenesis and branching from retinal neurons are regulated by the Rac1B/Rac3 GTPase. We have identified a functional connection between ADP-ribosylation factor (Arf) 6 and p95-APP1 during the regulation of Rac1B-mediated neuritogenesis. P95-APP1 is an ADP-ribosylation factor GTPase-activating protein (ArfGAP) of the GIT family expressed in the developing nervous system. We show that Arf6 has a predominant role in neurite extension compared with Arf1 and Arf5. Cotransfection experiments indicate a specific and cooperative potentiation of neurite extension by Arf6 and the carboxy-terminal portion of p95-APP1. Localization studies in neurons expressing different p95-derived constructs show a codistribution of p95-APP1 with Arf6, but not Arf1. Moreover, p95-APP1–derived proteins with a mutated or deleted ArfGAP domain prevent Rac1B-induced neuritogenesis, leading to PIX-mediated accumulation at large Rab11-positive endocytic vesicles. Our data support a role of p95-APP1 as a specific regulator of Arf6 in the control of membrane trafficking during neuritogenesis.
Cooperative Inhibition of Bone Morphogenetic Protein Signaling by Smurf1 and Inhibitory Smads
Authors: Murakami Gyo , Watabe Tetsuro , Takaoka Kunio , Miyazono Kohei , Imamura Takeshi
Published: April 2003

Smad ubiquitin regulatory factor (Smurf) 1 binds to receptor-regulated Smads for bone morphogenetic proteins (BMPs) Smad1/5 and promotes their degradation. In addition, Smurf1 associates with transforming growth factor-β type I receptor through the inhibitory Smad (I-Smad) Smad7 and induces their degradation. Herein, we examined whether Smurf1 negatively regulates BMP signaling together with the I-Smads Smad6/7. Smurf1 and Smad6 cooperatively induced secondary axes in Xenopus embryos. Using a BMP-responsive promoter-reporter construct in mammalian cells, we found that Smurf1 cooperated with I-Smad in inhibiting BMP signaling and that the inhibitory activity of Smurf1 was not necessarily correlated with its ability to bind to Smad1/5 directly. Smurf1 bound to BMP type I receptors via I-Smads and induced ubiquitination and degradation of these receptors. Moreover, Smurf1 associated with Smad1/5 indirectly through I-Smads and induced their ubiquitination and degradation. Smurf1 thus controls BMP signaling with and without I-Smads through multiple mechanisms.
Catabolite Degradation of Fructose-1,6-bisphosphatase in the YeastSaccharomyces cerevisiae: A Genome-wide Screen Identifies Eight NovelGIDGenes and Indicates the Existence of Two Degradation Pathways
Authors: Regelmann Jochen , Schüle Thomas , Josupeit Frank S. , Horak Jaroslav , Rose Matthias , Entian Karl-Dieter , Thumm Michael , Wolf Dieter H.
Published: April 2003

Metabolic adaptation of Saccharomyces cerevisiaecells from a nonfermentable carbon source to glucose induces selective, rapid breakdown of the gluconeogenetic key enzyme fructose-1,6-bisphosphatase (FBPase), a process called catabolite degradation. Herein, we identify eight novel GID genes required for proteasome-dependent catabolite degradation of FBPase. Four yeast proteins contain the CTLH domain of unknown function. All of them are Gid proteins. The site of catabolite degradation has been controversial until now. Two FBPase degradation pathways have been described, one dependent on the cytosolic ubiquitin-proteasome machinery, and the other dependent on vacuolar proteolysis. Interestingly, three of the novel Gid proteins involved in ubiquitin-proteasome–dependent degradation have also been reported by others to affect the vacuolar degradation pathway. As shown herein, additional genes suggested to be essential for vacuolar degradation are unnecessary for proteasome-dependent degradation. These data raise the question as to whether two FBPase degradation pathways exist that share components. Detailed characterization of Gid2p demonstrates that it is part of a soluble, cytosolic protein complex of at least 600 kDa. Gid2p is necessary for FBPase ubiquitination. Our studies have not revealed any involvement of vesicular intermediates in proteasome-dependent FBPase degradation. The influence of Ubp14p, a deubiquitinating enzyme, on proteasome-dependent catabolite degradation was further uncovered.
The Secretory Apparatus of an Ancient Eukaryote: Protein Sorting to Separate Export Pathways Occurs Before Formation of Transient Golgi-like Compartments
Authors: Marti Matthias , Li Yajie , Schraner Elisabeth M. , Wild Peter , Köhler Peter , Hehl Adrian B.
Published: April 2003

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.
Two Sets of Interacting Collagens Form Functionally Distinct Substructures within aCaenorhabditis elegansExtracellular Matrix
Authors: McMahon Laura , Muriel Joaquin M. , Roberts Brett , Quinn Martyn , Johnstone Iain L.
Published: April 2003

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of ∼154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed ∼2 h apart during matrix synthesis.
Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis
Authors: Zamanian Jennifer L. , Kelly Regis B.
Published: April 2003

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.
Crk Associates with a Multimolecular Paxillin/GIT2/β-PIX Complex and Promotes Rac-dependent Relocalization of Paxillin to Focal Contacts
Authors: Lamorte Louie , Rodrigues Sonia , Sangwan Veena , Turner Christopher E. , Park Morag
Published: April 2003

We have previously demonstrated that the CrkII and CrkL adapter proteins are required for the spreading of epithelial colonies and the breakdown of adherens junctions in response to hepatocyte growth factor. When overexpressed, CrkII and CrkL promote lamellipodia formation, cell spreading, and the loss of epithelial adherens junctions in the absence of hepatocyte growth factor. The exact mechanism by which Crk proteins elicit these changes is unclear. We show that the overexpression of CrkII or CrkL, but not Src homology 2 or amino-terminal Src homology 3 domain mutant Crk proteins, promotes the relocalization of Paxillin to focal contacts throughout the cell and within lamellipodia in a Rac-dependent manner. In stable cell lines overexpressing CrkII, enhanced lamellipodia formation and cell spreading correlate with an increased association of CrkII with Paxillin, GIT2 (an ARF-GAP) and β-PIX (a Rac1 exchange factor). Mutants of Paxillin that fail to associate with Crk or GIT2, or do not target to focal adhesions inhibit Crk-dependent cell spreading and lamellipodia formation. We conclude from these studies that the association of Crk with Paxillin is important for the spreading of epithelial colonies, by influencing the recruitment of Paxillin to focal complexes and promoting the enhanced assembly of Paxillin/GIT2/β-PIX complexes.
Role of Microtubules in Fusion of Post-Golgi Vesicles to the Plasma Membrane
Authors: Schmoranzer Jan , Simon Sanford M.
Published: April 2003

Biosynthetic cargo is transported away from the Golgi in vesicles via microtubules. In the cell periphery the vesicles are believed to engage actin and then dock to fusion sites at the plasma membrane. Using dual-color total internal reflection fluorescence microscopy, we observed that microtubules extended within 100 nm of the plasma membrane and post-Golgi vesicles remained on microtubules up to the plasma membrane, even as fusion to the plasma membrane initiated. Disruption of microtubules eliminated the tubular shapes of the vesicles and altered the fusion events: vesicles required multiple fusions to deliver all of their membrane cargo to the plasma membrane. In contrast, the effects of disrupting actin on fusion behavior were subtle. We conclude that microtubules, rather than actin filaments, are the cytoskeletal elements on which post-Golgi vesicles are transported until they fuse to the plasma membrane.
A GFP-based System to Uncouple mRNA Transport from Translation in a Single Living Neuron
Authors: Macchi Paolo , Hemraj Indradeo , Goetze Bernhard , Grunewald Barbara , Mallardo Massimo , Kiebler Michael A.
Published: April 2003

An inducible fluorescent system based on GFP is presented that allows for the uncoupling of dendritic mRNA transport from subsequent protein synthesis at the single cell level. The iron-responsive element (IRE) derived from ferritin mRNA in the 5′-UTR of the GFP reporter mRNA renders translation of its mRNA dependent on iron. The addition of the full-length 3′-UTR of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) after the stop codon of the GFP reading frame targets the reporter mRNA to dendrites of transfected fully polarized hippocampal neurons. As we show by time-lapse videomicroscopy, iron specifically turns on GFP reporter protein synthesis in a single transfected hippocampal neuron. We investigate whether GFP expression is affected—in addition to iron—by synaptic activity. Interestingly, synaptic activity has a clear stimulatory effect. Most importantly, however, this activity-dependent protein synthesis is critically dependent on the presence of the full-length 3′-UTR of CaMKIIα confirming that this sequence contains translational activation signals. The IRE-based system represents a new convenient tool to study local protein synthesis in mammalian cells where mRNA localization to a specific intracellular compartment occurs.
Accumulation of Cytoplasmic Dynein and Dynactin at Microtubule Plus Ends inAspergillus nidulansIs Kinesin Dependent
Authors: Zhang Jun , Li Shihe , Fischer Reinhard , Xiang Xin
Published: April 2003

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end–directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.
Lectin-like Ox-LDL Receptor Is Expressed in Human INT-407 Intestinal Cells: Involvement in the Transcytosis of Pancreatic Bile Salt–dependent Lipase
Authors: Bruneau Nadine , Richard Stéphane , Silvy Françoise , Verine Alain , Lombardo Dominique
Published: April 2003

We have recently shown that the pancreatic bile salt–dependent lipase (BSDL) can be taken up by intestinal cells and transported to the blood circulation. This mechanism likely involves (specific) receptor(s) able to bind BSDL and located at the apical intestinal cell membrane. In this study, using Int407 human intestinal cells cultured to form a tight epithelium, we attempted to characterize (the) BSDL receptor(s). We found that an apical 50-kDa protein was able to bind BSDL. Further, we have demonstrated that Int407 cells expressed the lectin-like oxidized-LDL receptor (LOX-1), the upregulation of which by oxidized-LDL potentiates the transcytosis of BSDL, whereas carrageenan and to a lesser extent polyinosinic acid and fucoidan decrease the enzyme transcytosis. The mAb JTX92, which blocks the LOX-1 receptor function, also impaired the BSDL transcytosis. To confirm these results, the cDNA encoding the human intestinal receptor LOX-1 has been cloned, inserted into vectors, and transfected into Int407 cells. Overexpression of LOX-1 by these cells leads to a substantial increase in the BSDL transcytosis. Globally, these data support the view that LOX-1 could be an intestinal receptor for BSDL, which is implicated in the transcytosis of this enzyme throughout Int407 cells.
Nestin Promotes the Phosphorylation-dependent Disassembly of Vimentin Intermediate Filaments During Mitosis
Authors: Chou Ying-Hao , Khuon Satya , Herrmann Harald , Goldman Robert D.
Published: April 2003

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase–promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.
Stress-induced Gene Expression inCandida albicans: Absence of a General Stress Response
Authors: Enjalbert Brice , Nantel André , Whiteway Malcolm
Published: April 2003

We used transcriptional profiling to investigate the response of the fungal pathogen Candida albicans to temperature and osmotic and oxidative stresses under conditions that permitted >60% survival of the challenged cells. Each stress generated the transient induction of a specific set of genes including classic markers observed in the stress responses of other organisms. We noted that the classical hallmarks of the general stress response observed inSaccharomyces cerevisiae are absent from C. albicans; no C. albicans genes were significantly induced in a common response to the three stresses. This observation is supported by our inability to detect stress cross-protection in C. albicans. Similarly, in C. albicans there is essentially no induction of carbohydrate reserves like glycogen and trehalose in response to a mild stress, unlike the situation in S. cerevisiae. Thus C. albicans lacks the strong general stress response exhibited byS. cerevisiae.
A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium
Authors: Sun Binggang , Firtel Richard A.
Published: April 2003

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.
Endosomal Dynamics of Met Determine Signaling Output
Authors: Hammond Dean E. , Carter Stephanie , McCullough John , Urbé Sylvie , Vande Woude George , Clague Michael J.
Published: April 2003

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place ( Hammond et al., 2001 ). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5–10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.
A Kinesin Mutant with an Atypical Bipolar Spindle Undergoes Normal Mitosis
Authors: Marcus A. I. , Li W. , Ma H. , Cyr R. J.
Published: April 2003

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.
Requirement for Down-Regulation of the CCAAT-binding Activity of the NF-Y Transcription Factor during Skeletal Muscle Differentiation
Authors: Gurtner Aymone , Manni Isabella , Fuschi Paola , Mantovani Roberto , Guadagni Fiorella , Sacchi Ada , Piaggio Giulia
Published: April 2003

NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC, all required for DNA binding. All subunits are expressed in proliferating skeletal muscle cells, whereas NF-YA alone is undetectable in terminally differentiated cells in vitro. By immunohistochemistry, we show that the NF-YA protein is not expressed in the nuclei of skeletal and cardiac muscle cells in vivo. By chromatin immunoprecipitation experiments, we demonstrate herein that NF-Y does not bind to the CCAAT boxes of target promoters in differentiated muscle cells. Consistent with this, the activity of these promoters is down-regulated in differentiated muscle cells. Finally, forced expression of the NF-YA protein in cells committed to differentiate leads to an impairment in the down-regulation of cyclin A, cyclin B1, and cdk1 expression and is accompanied by a delay in myogenin expression. Thus, our results indicate that the suppression of NF-Y function is of crucial importance for the inhibition of several cell cycle genes and the induction of the early muscle-specific program in postmitotic muscle cells.
An Early Function during Transcription for the Yeast mRNA Export Factor Dbp5p/Rat8p Suggested by Its Genetic and Physical Interactions with Transcription Factor IIH Components
Authors: Estruch Francisco , Cole Charles N.
Published: April 2003

The yeast DEAD-box protein Dbp5p/Rat8p is an essential factor for mRNA export and shuttles between the nucleus and the cytoplasm. It is concentrated at the cytoplasmic fibrils of the nuclear pore complex where it interacts with several nucleoporins. On the basis of this localization, it has been suggested that it might participate in a terminal step of RNA export, the release from the mRNA of proteins that accompany the mRNA during translocation through nuclear pores. In this report, we present evidence linking Dbp5p to transcription. Two different screens identified genetic interactions between DBP5 and genes involved in early transcription events, initiation and promoter clearance. Mutations of transcription proteins expected to impair transcription act as suppressors ofdbp5 mutants, whereas those that may act to increase transcription are synthetically lethal with dbp5mutations. We also show that growth and mRNA export indbp5 mutant strains are dependent on the carboxy-terminal domain of the RNA pol II largest subunit. Finally, we show that Dbp5p associates physically with components of transcription factor IIH. Because these interactions affect not only growth but also mRNA export, they are likely to reflect a functional relationship between Dbp5p and the transcription machinery. Together, our results suggest a nuclear role for Dbp5 during the early steps of transcription.
Functional Significance of the Specific Sites Phosphorylated in Desmin at Cleavage Furrow: Aurora-B May Phosphorylate and Regulate Type III Intermediate Filaments during Cytokinesis Coordinatedly with Rho-kinase
Authors: Kawajiri Aie , Yasui Yoshihiro , Goto Hidemasa , Tatsuka Masaaki , Takahashi Masahide , Nagata Koh-ichi , Inagaki Masaki
Published: April 2003

Aurora-B is a protein kinase required for chromosome segregation and the progression of cytokinesis during the cell cycle. We report here that Aurora-B phosphorylates GFAP and desmin in vitro, and this phosphorylation leads to a reduction in filament forming ability. The sites phosphorylated by Aurora-B; Thr-7/Ser-13/Ser-38 of GFAP, and Thr-16 of desmin are common with those related to Rho-associated kinase (Rho-kinase), which has been reported to phosphorylate GFAP and desmin at cleavage furrow during cytokinesis. We identified Ser-59 of desmin to be a specific site phosphorylated by Aurora-B in vitro. Use of an antibody that specifically recognized desmin phosphorylated at Ser-59 led to the finding that the site is also phosphorylated specifically at the cleavage furrow during cytokinesis in Saos-2 cells. Desmin mutants, in which in vitro phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defects in filament separation between daughter cells in cytokinesis. The results presented here suggest the possibility that Aurora-B may regulate cleavage furrow-specific phosphorylation and segregation of type III IFs coordinatedly with Rho-kinase during cytokinesis.
Regulation of the Yeast Amphiphysin Homologue Rvs167p by Phosphorylation
Authors: Friesen Helena , Murphy Kelly , Breitkreutz Ashton , Tyers Mike , Andrews Brenda
Published: April 2003

The yeast amphiphysin homologue Rvs167p plays a role in regulation of the actin cytoskeleton, endocytosis, and sporulation. Rvs167p is a phosphoprotein in vegetatively growing cells and shows increased phosphorylation upon treatment with mating pheromone. Previous work has shown that Rvs167p can be phosphorylated in vitro by the cyclin-dependent kinase Pho85p complexed with its cyclin Pcl2p. Using chymotryptic phosphopeptide mapping, we have identified the sites on which Rvs167p is phosphorylated in vitro by Pcl2p-Pho85p. We have shown that these same sites are phosphorylated in vivo during vegetative growth and that phosphorylation at two of these sites is Pcl-Pho85p dependent. In cells treated with mating pheromone, the MAP kinase Fus3p is needed for full phosphorylation of Rvs167p. Functional genomics and genetics experiments revealed that mutation of other actin cytoskeleton genes compromises growth of a strain in which phosphorylation of Rvs167p is blocked by mutation. Phosphorylation of Rvs167p inhibits its interaction in vitro with Las17p, an activator of the Arp2/3 complex, as well as with a novel protein, Ymr192p. Our results suggest that phosphorylation of Rvs167p by a cyclin-dependent kinase and by a MAP kinase is an important mechanism for regulating protein complexes involved in actin cytoskeleton function.
Regulation of Sodium Channel Activity by Capping of Actin Filaments
Authors: Shumilina Ekaterina V. , Negulyaev Yuri A. , Morachevskaya Elena A. , Hinssen Horst , Khaitlina Sofia Yu
Published: April 2003

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.
p250GAP, a Novel Brain-enriched GTPase-activating Protein for Rho Family GTPases, Is Involved in theN-Methyl-d-aspartate Receptor Signaling
Authors: Nakazawa Takanobu , Watabe Ayako M. , Tezuka Tohru , Yoshida Yutaka , Yokoyama Kazumasa , Umemori Hisashi , Inoue Akihiro , Okabe Shigeo , Manabe Toshiya , Yamamoto Tadashi
Published: April 2003

N-Methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRϵ2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRϵ2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.
Zwilch, a New Component of the ZW10/ROD Complex Required for Kinetochore Functions
Authors: Williams Byron C. , Li ZeXiao , Liu Songtao , Williams Erika V. , Leung Garmay , Yen Tim J. , Goldberg Michael L.
Published: April 2003

The Zeste-White 10 (ZW10) and Rough Deal (ROD) proteins are part of a complex necessary for accurate chromosome segregation. This complex recruits cytoplasmic dynein to the kinetochore and participates in the spindle checkpoint. We used immunoaffinity chromatography and mass spectroscopy to identify theDrosophila proteins in this complex. We found that the complex contains an additional protein we name Zwilch. Zwilch localizes to kinetochores and kinetochore microtubules in a manner identical to ZW10 and ROD. We have also isolated azwilch mutant, which exhibits the same mitotic phenotypes associated with zw10 and rodmutations: lagging chromosomes at anaphase and precocious sister chromatid separation upon activation of the spindle checkpoint. Zwilch's role within the context of this complex is evolutionarily conserved. The human Zwilch protein (hZwilch) coimmunoprecipitates with hZW10 and hROD from HeLa cell extracts and localizes to the kinetochores at prometaphase. Finally, we discuss immunoaffinity chromatography results that suggest the existence of a weak interaction between the ZW10/ROD/Zwilch complex and the kinesin-like kinetochore component CENP-meta.
Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant
Authors: Tanaka Yoshinari , Nakanishi Hiroyuki , Kakunaga Shigeki , Okabe Noriko , Kawakatsu Tomomi , Shimizu Kazuya , Takai Yoshimi
Published: April 2003

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.
MTG1 Codes for a Conserved Protein Required for Mitochondrial Translation
Authors: Barrientos Antoni , Korr Daniel , Barwell Karen J. , Sjulsen Christian , Gajewski Carl D. , Manfredi Giovanni , Ackerman Sharon , Tzagoloff Alexander
Published: April 2003

The MTG1 gene of Saccharomyces cerevisiae, corresponding to ORF YMR097c on chromosome XIII, codes for a mitochondrial protein essential for respiratory competence. A human homologue of Mtg1p capable of partially rescuing the respiratory deficiency of a yeast mtg1 mutant has also been localized in mitochondria. Mtg1p is a member of a family of GTPases with largely unknown functions. The respiratory deficiency of mtg1 mutants stems from a defect in mitochondrial protein synthesis. Mutations in the 21S rRNA locus are able to suppress the translation defect of mtg1 null mutants. This points to the 21S rRNA or the large ribosomal subunit as the most likely target of Mtg1p action. The presence of mature size 15S and 21S mitochondrial rRNAs in mtg1 mutants excludes Mtg1p from being involved in transcription or processing of these RNAs. More likely, Mtg1p functions in assembly of the large ribosomal subunit. This is consistent with the peripheral localization of Mtg1p on the matrix side of the inner membrane and the results of in vivo mitochondrial translation assays in a temperature-sensitive mtg1 mutant.
Intercellular Organelle Traffic through Cytoplasmic Bridges in Early Spermatids of the Rat: Mechanisms of Haploid Gene Product Sharing
Authors: Ventelä Sami , Toppari Jorma , Parvinen Martti
Published: April 2003

Stable cytoplasmic bridges (or ring canals) connecting the clone of spermatids are assumed to facilitate the sharing of haploid gene products and synchronous development of the cells. We have visualized these cytoplasmic bridges under phase-contrast optics and recorded the sharing of cytoplasmic material between the spermatids by a digital time-lapse imaging system ex vivo. A multitude of small (ca. 0.5 μm) granules were seen to move continuously over the bridges, but only 28% of those entering the bridge were actually transported into other cell. The average speed of the granules decreased significantly during the passage. Immunocytochemistry revealed that some of the shared granules contained haploid cell-specific gene product TRA54. We also demonstrate the novel function for the Golgi complex in acrosome system formation by showing that TRA54 is processed in Golgi complex and is transported into acrosome system of neighboring spermatid. In addition, we propose an intercellular transport function for the male germ cell-specific organelle chromatoid body. This mRNA containing organelle, ca. 1.8 μm in diameter, was demonstrated to go over the cytoplasmic bridge from one spermatid to another. Microtubule inhibitors prevented all organelle movements through the bridges and caused a disintegration of the chromatoid body. This is the first direct demonstration of an organelle traffic through cytoplasmic bridges in mammalian spermatogenesis. Golgi-derived haploid gene products are shared between spermatids, and an active involvement of the chromatoid body in intercellular material transport between round spermatids is proposed.
Vps51p Mediates the Association of the GARP (Vps52/53/54) Complex with the Late Golgi t-SNARE Tlg1p
Authors: Conibear Elizabeth , Cleck Jessica N. , Stevens Tom H.
Published: April 2003

Multisubunit tethering complexes may contribute to the specificity of membrane fusion events by linking transport vesicles to their target membrane in an initial recognition event that promotes SNARE assembly. However, the interactions that link tethering factors to the other components of the vesicle fusion machinery are still largely unknown. We have previously identified three subunits of a Golgi-localized complex (the Vps52/53/54 complex) that is required for retrograde transport to the late Golgi. This complex interacts with a Rab and a SNARE protein found at the late Golgi and is related to two other multisubunit tethering complexes: the COG complex and the exocyst. Here we show that the Vps52/53/54 complex has an additional subunit, Vps51p. All four members of this tetrameric GARP (Golgi-associated retrograde protein) complex are required for two distinct retrograde transport pathways, from both early and late endosomes, back to the TGN.vps51 mutants exhibit a distinct phenotype suggestive of a regulatory role. Indeed, we find that Vps51p mediates the interaction between Vps52/53/54 and the t-SNARE Tlg1p. The binding of this small, coiled-coil protein to the conserved N-terminal domain of the t-SNARE therefore provides a crucial link between components of the tethering and the fusion machinery.
Fission Yeast cdc31p Is a Component of the Half-bridge and Controls SPB Duplication
Authors: Paoletti Anne , Bordes Nicole , Haddad Raphaël , Schwartz Cindi L. , Chang Fred , Bornens Michel
Published: April 2003

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461–1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Δcdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.
The Third P-loop Domain in Cytoplasmic Dynein Heavy Chain Is Essential for Dynein Motor Function and ATP-sensitive Microtubule Binding
Authors: Silvanovich Andre , Li Min-gang , Serr Madeline , Mische Sarah , Hays Thomas S.
Published: April 2003

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of theDrosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.
The Synaptojanin-like Protein Inp53/Sjl3 Functions with Clathrin in a Yeast TGN-to-Endosome Pathway Distinct from the GGA Protein-dependent Pathway
Authors: Ha Seon-Ah , Torabinejad Javad , DeWald Daryll B. , Wenk Markus R. , Lucast Louise , De Camilli Pietro , Newitt Richard A. , Aebersold Ruedi , Nothwehr Steven F.
Published: April 2003

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate “slow delivery” of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.
Procyclin Null Mutants ofTrypanosoma bruceiExpress Free Glycosylphosphatidylinositols on Their Surface
Authors: Vassella Erik , Bütikofer Peter , Engstler Markus , Jelk Jennifer , Roditi Isabel
Published: April 2003

Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.