bluuebunny/crossref_metadata_2025 huggingface dataset:
Rotational Coupling of the Transmembrane and Kinase Domains of the Neu Receptor Tyrosine Kinase
Authors: Bell Charlotte A. , Tynan John A. , Hart Kristen C. , Meyer April N. , Robertson Scott C. , Donoghue Daniel J.
Published: July 2013

Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV]n, previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-β suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.
ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane
Authors: Joyce Charles W. , Shelness Gregory S. , Davis Matthew A. , Lee Richard G. , Skinner Kelly , Anderson Richard A. , Rudel Lawrence L.
Published: July 2013

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.
Testing the 3Q:1R “Rule”: Mutational Analysis of the Ionic “Zero” Layer in the Yeast Exocytic SNARE Complex Reveals No Requirement for Arginine
Authors: Katz Luba , Brennwald Patrick
Published: July 2013

The crystal structure of the synaptic SNARE complex reveals a parallel four-helix coiled-coil arrangement; buried in the hydrophobic core of the complex is an unusual ionic layer composed of three glutamines and one arginine, each provided by a separate α-helix. The presence of glutamine or arginine residues in this position is highly conserved across the t- and v-SNARE families, and it was recently suggested that a 3Q:1R ratio is likely to be a general feature common to all SNARE complexes. In this study, we have used genetic and biochemical assays to test this prediction with the yeast exocytic SNARE complex. We have determined that the relative position of Qs and Rs within the layer is not critical for biological activity and that Q-to-R substitutions in the layer reduce complex stability and result in lethal or conditional lethal growth defects. Surprisingly, SNARE complexes composed of four glutamines are fully functional for assembly in vitro and exocytic function in vivo. We conclude that the 3Q:1R layer composition is not required within the yeast exocytic SNARE complex because complexes containing four Q residues in the ionic layer appear by all criteria to be functionally equivalent. The unexpected flexibility of this layer suggests that there is no strict requirement for the 3Q:1R combination and that the SNARE complexes at other stages of transport may be composed entirely of Q-SNAREs or other noncanonical combinations.
The Yeast Nucleoporin Nup53p Specifically Interacts with Nic96p and Is Directly Involved in Nuclear Protein Import
Authors: Fahrenkrog Birthe , Hübner Wolfgang , Mandinova Anna , Panté Nelly , Keller Walter , Aebi Ueli
Published: July 2013

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ∼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 withNIC96 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.
Asnc1Endocytosis Mutant: Phenotypic Analysis and Suppression by Overproduction of Dihydrosphingosine Phosphate Lyase
Authors: Grote Eric , Vlacich Greg , Pypaert Marc , Novick Peter J.
Published: July 2013

The v-SNARE proteins Snc1p and Snc2p are required for fusion of secretory vesicles with the plasma membrane in yeast. Mutation of a methionine-based sorting signal in the cytoplasmic domain of either Sncp inhibits Sncp endocytosis and prevents recycling of Sncp to the Golgi after exocytosis. snc1-M43A mutant yeast have reduced growth and secretion rates and accumulate post-Golgi secretory vesicles and fragmented vacuoles. However, cells continue to grow and secrete for several hours after de novo Snc2-M42A synthesis is repressed. DPL1, the structural gene for dihydrosphingosine phosphate lyase, was selected as a high copy numbersnc1-M43A suppressor. Because DPL1 also partially suppresses the growth and secretion phenotypes of asnc deletion, we propose that enhanced degradation of dihydrosphingosine-1-phosphate allows an alternative protein to replace Sncp as the secretory vesicle v-SNARE.
Complex Phenotype of Mice Lacking Occludin, a Component of Tight Junction Strands
Authors: Saitou Mitinori , Furuse Mikio , Sasaki Hiroyuki , Schulzke Jörg-Dieter , Fromm Michael , Takano Hiroshi , Noda Tetsuo , Tsukita Shoichiro
Published: July 2013

Occludin is an integral membrane protein with four transmembrane domains that is exclusively localized at tight junction (TJ) strands. Here, we describe the generation and analysis of mice carrying a null mutation in the occludin gene. Occludin −/− mice were born with no gross phenotype in the expected Mendelian ratios, but they showed significant postnatal growth retardation. Occludin −/− males produced no litters with wild-type females, whereas occludin −/− females produced litters normally when mated with wild-type males but did not suckle them. In occludin −/− mice, TJs themselves did not appear to be affected morphologically, and the barrier function of intestinal epithelium was normal as far as examined electrophysiologically. However, histological abnormalities were found in several tissues, i.e., chronic inflammation and hyperplasia of the gastric epithelium, calcification in the brain, testicular atrophy, loss of cytoplasmic granules in striated duct cells of the salivary gland, and thinning of the compact bone. These phenotypes suggested that the functions of TJs as well as occludin are more complex than previously supposed.
Golgi Apparatus Immunolocalization of Endomannosidase Suggests Post-Endoplasmic Reticulum Glucose Trimming: Implications for Quality Control
Authors: Zuber Christian , Spiro Mary Jane , Guhl Bruno , Spiro Robert G. , Roth Jürgen
Published: July 2013

Trimming of N-linked oligosaccharides by endoplasmic reticulum (ER) glucosidase II is implicated in quality control of protein folding. An alternate glucosidase II-independent deglucosylation pathway exists, in which endo-α-mannosidase cleaves internally the glucose-substituted mannose residue of oligosaccharides. By immunogold labeling, we detected most endomannosidase in cis/medial Golgi cisternae (83.8% of immunogold labeling) and less in the intermediate compartment (15.1%), but none in the trans-Golgi apparatus and ER, including its transitional elements. This dual localization became more pronounced under 15°C conditions indicative of two endomannosidase locations. Under experimental conditions when the intermediate compartment marker p58 was retained in peripheral sites, endomannosidase was redistributed to the Golgi apparatus. Double immunogold labeling established a mutually exclusive distribution of endomannosidase and glucosidase II, whereas calreticulin was observed in endomannosidase-reactive sites (17.3% in intermediate compartment, 5.7% in Golgi apparatus) in addition to the ER (77%). Our results demonstrate that glucose trimming of N-linked oligosaccharides is not limited to the ER and that protein deglucosylation by endomannosidase in the Golgi apparatus and intermediate compartment additionally ensures that processing to mature oligosaccharides can continue. Thus, endomannosidase localization suggests that a quality control ofN-glycosylation exists in the Golgi apparatus.
The Latent Transforming Growth Factor-β–binding Protein-1 Promotes In Vitro Differentiation of Embryonic Stem Cells into Endothelium
Authors: Gualandris Anna , Annes Justin P. , Arese Marco , Noguera Irene , Jurukovski Vladimir , Rifkin Daniel B.
Published: July 2013

The latent transforming growth factor-β–binding protein-1 (LTBP-1) belongs to a family of extracellular glycoproteins that includes three additional isoforms (LTBP-2, -3, and -4) and the matrix proteins fibrillin-1 and -2. Originally described as a TGF-β–masking protein, LTBP-1 is involved both in the sequestration of latent TGF-β in the extracellular matrix and the regulation of its activation in the extracellular environment. Whereas the expression of LTBP-1 has been analyzed in normal and malignant cells and rodent and human tissues, little is known about LTBP-1 in embryonic development. To address this question, we used murine embryonic stem (ES) cells to analyze the appearance and role of LTBP-1 during ES cell differentiation. In vitro, ES cells aggregate to form embryoid bodies (EBs), which differentiate into multiple cell lineages. We analyzed LTBP-1 gene expression and LTBP-1 fiber appearance with respect to the emergence and distribution of cell types in differentiating EBs. LTBP-1 expression increased during the first 12 d in culture, appeared to remain constant between d 12 and 24, and declined thereafter. By immunostaining, fibrillar LTBP-1 was observed in those regions of the culture containing endothelial, smooth muscle, and epithelial cells. We found that inclusion of a polyclonal antibody to LTBP-1 during EB differentiation suppressed the expression of the endothelial specific genes ICAM-2 and von Willebrand factor and delayed the organization of differentiated endothelial cells into cord-like structures within the growing EBs. The same effect was observed when cultures were treated with either antibodies to TGF-β or the latency associated peptide, which neutralize TGF-β. Conversely, the organization of endothelial cells was enhanced by incubation with TGF-β1. These results suggest that during differentiation of ES cells LTBP-1 facilitates endothelial cell organization via a TGF-β–dependent mechanism.
New Components of a System for Phosphate Accumulation and Polyphosphate Metabolism inSaccharomyces cerevisiaeRevealed by Genomic Expression Analysis
Authors: Ogawa Nobuo , DeRisi Joseph , Brown Patrick O.
Published: July 2013

The PHO regulatory pathway is involved in the acquisition of phosphate (Pi) in the yeast Saccharomyces cerevisiae. When extracellular Piconcentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified. The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site. Eight of these genes, PHM1–PHM8, had no previously defined function in phosphate metabolism. The amino acid sequences ofPHM1 (YFL004w), PHM2 (YPL019c),PHM3 (YJL012c), and PHM4 (YER072w) are 32–56% identical. The phm3 and phm4single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and Pi. The phenotype of thephm5 mutant suggests that PHM5 (YDR452w)is essential for normal catabolism of polyP in the yeast vacuole. Taken together, the results reveal important new features of a genetic system that plays a critical role in Piacquisition and polyP metabolism in yeast.
A Decrease in Membrane Tension Precedes Successful Cell-Membrane Repair
Authors: Togo Tatsuru , Krasieva Tatiana B. , Steinhardt Richard A.
Published: July 2013

We hypothesized that the requirement for Ca2+-dependent exocytosis in cell-membrane repair is to provide an adequate lowering of membrane tension to permit membrane resealing. We used laser tweezers to form membrane tethers and measured the force of those tethers to estimate the membrane tension of Swiss 3T3 fibroblasts after membrane disruption and during resealing. These measurements show that, for fibroblasts wounded in normal Ca2+Ringer's solution, the membrane tension decreased dramatically after the wounding and resealing coincided with a decrease of ∼60% of control tether force values. However, the tension did not decrease if cells were wounded in a low Ca2+Ringer's solution that inhibited both membrane resealing and exocytosis. When cells were wounded twice in normal Ca2+Ringer's solution, decreases in tension at the second wound were 2.3 times faster than at the first wound, correlating well with twofold faster resealing rates for repeated wounds. The facilitated resealing to a second wound requires a new vesicle pool, which is generated via a protein kinase C (PKC)-dependent and brefeldin A (BFA)-sensitive process. Tension decrease at the second wound was slowed or inhibited by PKC inhibitor or BFA. Lowering membrane tension by cytochalasin D treatment could substitute for exocytosis and could restore membrane resealing in low Ca2+Ringer's solution.
Nuclear pre-mRNA Compartmentalization: Trafficking of Released Transcripts to Splicing Factor Reservoirs
Authors: Melčák Ivo , Cermanová Štěpánka , Jirsová Kateřina , Koberna Karel , Malı́nský Jan , Raška Ivan
Published: July 2013

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein–Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.
An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast
Authors: Luo Wen-jie , Chang Amy
Published: July 2013

The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Severalvps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, andvps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 andvps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (andvps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface invps1 cells. We hypothesize that in vps8and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway.
Occludin 1B, a Variant of the Tight Junction Protein Occludin
Authors: Muresan Zoia , Paul David L. , Goodenough Daniel A.
Published: July 2013

Occludin and claudin are the major integral membrane components of the mammalian tight junction. Although more than 11 distinct claudins have been identified, only 1 occludin transcript has been reported thus far. Therefore, we searched by reverse transcription–PCR for occludin-related sequences in Madin–Darby canine kidney (MDCK) mRNA and identified a transcript encoding an alternatively spliced form of occludin, designated occludin 1B. The occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Analysis of the MDCK occludin gene revealed an exon containing the 193-base pair sequence between the exons encoding the original N terminus and the distal sequence, suggesting that occludin and occludin 1B arise from alternative splicing of one transcript. To assess the expression and distribution of occludin 1B, an antibody was raised against its unique N-terminal domain. Immunolabeling of occludin 1B in MDCK cells revealed a distribution indistinguishable from that of occludin. Furthermore, occludin 1B staining at cell-to-cell contacts was also found in cultured T84 human colon carcinoma cells and in frozen sections of mouse intestine. Immunoblots of various mouse tissues revealed broad coexpression of occludin 1B with occludin. The wide epithelial distribution and the conservation across species suggests a potentially important role for occludin 1B in the structure and function of the tight junction.
The Role of the Tethering Proteins p115 and GM130 in Transport through the Golgi Apparatus In Vivo
Authors: Seemann Joachim , Jokitalo Eija Jämsä , Warren Graham
Published: July 2013

Biochemical data have shown that COPI-coated vesicles are tethered to Golgi membranes by a complex of at least three proteins: p115, giantin, and GM130. p115 binds to giantin on the vesicles and to GM130 on the membrane. We now examine the function of this tethering complex in vivo. Microinjection of an N-terminal peptide of GM130 or overexpression of GM130 lacking this N-terminal peptide inhibits the binding of p115 to Golgi membranes. Electron microscopic analysis of single microinjected cells shows that the number of COP-sized transport vesicles in the Golgi region increases substantially, suggesting that transport vesicles continue to bud but are less able to fuse. This was corroborated by quantitative immunofluorescence analysis, which showed that the intracellular transport of the VSV-G protein was significantly inhibited. Together, these data suggest that this tethering complex increases the efficiency with which transport vesicles fuse with their target membrane. They also provide support for a model of mitotic Golgi fragmentation in which the tethering complex is disrupted by mitotic phosphorylation of GM130.
The Yeast Kinesin-related Protein Smy1p Exerts Its Effects on the Class V Myosin Myo2p via a Physical Interaction
Authors: Beningo Karen A. , Lillie Sue H. , Brown Susan S.
Published: July 2013

We have discovered evidence for a physical interaction between a class V myosin, Myo2p, and a kinesin-related protein, Smy1p, in budding yeast. These proteins had previously been linked by genetic and colocalization studies, but we had been unable to determine the nature of their association. We now show by two-hybrid analysis that a 69-amino acid region of the Smy1p tail interacts with the globular portion of the Myo2p tail. Deletion of this myosin-binding region of Smy1p eliminates its ability to colocalize with Myo2p and to overcome the myo2–66 mutant defects, suggesting that the interaction is necessary for these functions. Further insights about the Smy1p–Myo2p interaction have come from studies of a new mutant allele, myo2–2, which causes a loss of Myo2p localization. We report that Smy1p localization is also lost in themyo2–2 mutant, demonstrating that Smy1p localization is dependent on Myo2p. We also found that overexpression of Smy1p partially restores myo2–2p localization in a myosin-binding region–dependent manner. Thus, overexpression of Smy1p can overcome defects in both the head and tail domains of Myo2p (caused by themyo2–66 and myo2–2 alleles, respectively). We propose that Smy1p enhances some aspect of Myo2p function, perhaps delivery or docking of vesicles at the bud tip.
Role of Ribosome and Translocon Complex during Folding of Influenza Hemagglutinin in the Endoplasmic Reticulum of Living Cells
Authors: Chen Wei , Helenius Ari
Published: July 2013

Protein folding in the living cell begins cotranslationally. To analyze how it is influenced by the ribosome and by the translocon complex during translocation into the endoplasmic reticulum, we expressed a mutant influenza hemagglutinin (a type I membrane glycoprotein) with a C-terminal extension. Analysis of the nascent chains by two-dimensional SDS-PAGE showed that ribosome attachment as such had little effect on ectodomain folding or trimer assembly. However, as long as the chains were ribosome bound andinside the translocon complex, formation of disulfides was partially suppressed, trimerization was inhibited, and the protein protected against aggregation.
Degradation of the Transcription Factor Gcn4 Requires the Kinase Pho85 and the SCFCDC4Ubiquitin–Ligase Complex
Authors: Meimoun Ariella , Holtzman Tsvi , Weissman Ziva , McBride Helen J. , Stillman David J. , Fink Gerald R. , Kornitzer Daniel
Published: July 2013

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCFCDC4activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.
The Lipid-anchored Ectodomain of Influenza Virus Hemagglutinin (GPI-HA) Is Capable of Inducing Nonenlarging Fusion Pores
Authors: Markosyan Ruben M. , Cohen Fredric S. , Melikyan Grigory B.
Published: July 2013

GPI-linked hemagglutinin (GPI-HA) of influenza virus was thought to induce hemifusion without pore formation. Cells expressing either HA or GPI-HA were bound to red blood cells, and their fusion was compared by patch-clamp capacitance measurements and fluorescence microscopy. It is now shown that under more optimal fusion conditions than have been used previously, GPI-HA is also able to induce fusion pore formation before lipid dye spread, although with fewer pores formed than those induced by HA. The GPI-HA pores did not enlarge substantially, as determined by the inability of a small aqueous dye to pass through them. The presence of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate or octadecylrhodamine B in red blood cells significantly increased the probability of pore formation by GPI-HA; the dyes affected pore formation to a much lesser degree for HA. This greater sensitivity of pore formation to lipid composition suggests that lipids are a more abundant component of a GPI-HA fusion pore than of an HA pore. The finding that GPI-HA can induce pores indicates that the ectodomain of HA is responsible for all steps up to the initial membrane merger and that the transmembrane domain, although not absolutely required, ensures reliable pore formation and is essential for pore growth. GPI-HA is the minimal unit identified to date that supports fusion to the point of pore formation.
A Screen for Dominant Negative Mutants ofSEC18Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis
Authors: Steel Gregor J. , Harley Carol , Boyd Alan , Morgan Alan
Published: July 2013

An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex ofN-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulatedSEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. Thesec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.
Nuclear Matrix-like Filaments and Fibrogranular Complexes Form through the Rearrangement of Specific Nuclear Ribonucleoproteins
Authors: Tan Jia-huai , Wooley John C. , LeStourgeon Wallace M.
Published: July 2013

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)3B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 μm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the “nuclear matrix.” Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.
The Kex2p Proregion Is Essential for the Biosynthesis of an Active Enzyme and Requires a C-terminal Basic Residue for Its Function
Authors: Lesage Guillaume , Prat Annik , Lacombe Julie , Thomas David Y. , Seidah Nabil G. , Boileau Guy
Published: July 2013

The Saccharomyces cerevisiae prohormone-processing enzyme Kex2p is biosynthesized as an inactive precursor extended by its N-terminal proregion. Here we show that deletion of the proregion renders Kex2p inactive both in vivo and in vitro. Absence of the proregion impaired glycosylation and stability and resulted in the retention of the enzyme in the endoplasmic reticulum. These phenotypes were partially complemented by expression of the proregion intrans. Trans complementation was specific to Kex2p proregion because expression of any of the seven mammalian prohormone convertase propeptides had no effect. These data are consistent with a model whereby Kex2p proregion functions as an intramolecular chaperone and indicate that covalent linkage to the protein is not an absolute requirement for proregion function. Furthermore, extensive mutagenesis revealed that, in addition to their function as proteolytic recognition sites, C-terminal basic residues play an active role in proregion-dependent Kex2p activation.
Proinsulin Endoproteolysis Confers Enhanced Targeting of Processed Insulin to the Regulated Secretory Pathway
Authors: Kuliawat Regina , Prabakaran Daniel , Arvan Peter
Published: July 2013

Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as “sorting receptors” to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.
Dynamic Regulation of Activated Leukocyte Cell Adhesion Molecule–mediated Homotypic Cell Adhesion through the Actin Cytoskeleton
Authors: Nelissen Judith M. D. T. , Peters Inge M. , de Grooth Bart G. , van Kooyk Yvette , Figdor Carl G.
Published: July 2013

Restricted expression of activated leukocyte cell adhesion molecule (ALCAM) by hematopoietic cells suggests an important role in the immune system and hematopoiesis. To get insight into the mechanisms that control ALCAM-mediated adhesion we have investigated homotypic ALCAM–ALCAM interactions. Here, we demonstrate that the cytoskeleton regulates ALCAM-mediated cell adhesion because inhibition of actin polymerization by cytochalasin D (CytD) strongly induces homotypic ALCAM–ALCAM interactions. This induction of cell adhesion is likely due to clustering of ALCAM at the cell surface, which is observed after CytD treatment. Single-particle tracking demonstrated that the lateral mobility of ALCAM in the cell membrane is increased 30-fold after CytD treatment. In contrast, both surface distribution and adhesion of a glycosylphosphatidylinositol (GPI)-anchored ALCAM mutant are insensitive to CytD, despite the increase in lateral mobility of GPI-ALCAM upon CytD treatment. This demonstrates that clustering of ALCAM is essential for cell adhesion, whereas enhanced diffusion of ALCAM alone is not sufficient for cluster formation. In addition, upon ligand binding, both free diffusion and the freely dragged distance of wild-type ALCAM, but not of GPI-ALCAM, are reduced over time, suggesting strengthening of the cytoskeleton linkage. From these findings we conclude that activation of ALCAM-mediated adhesion is dynamically regulated through actin cytoskeleton-dependent clustering.
Exo1 Roles for Repair of DNA Double-Strand Breaks and Meiotic Crossing Over inSaccharomyces cerevisiae
Authors: Tsubouchi Hideo , Ogawa Hideyuki
Published: July 2013

The MRE11, RAD50, andXRS2 genes of Saccharomyces cerevisiaeare involved in the repair of DNA double-strand breaks (DSBs) produced by ionizing radiation and by radiomimetic chemicals such as methyl methanesulfonate (MMS). In these mutants, single-strand DNA degradation in a 5′ to 3′ direction from DSB ends is reduced. Multiple copies of the EXO1 gene, encoding a 5′ to 3′ double-strand DNA exonuclease, were found to suppress the high MMS sensitivity of these mutants. The exo1 single mutant shows weak MMS sensitivity. When an exo1 mutation is combined with anmre11 mutation, both repair of MMS-induced damage and processing of DSBs are more severely reduced than in either single mutant, suggesting that Exo1 and Mre11 function independently in DSB processing. During meiosis, transcription of the EXO1gene is highly induced. In meiotic cells, the exo1mutation reduces the processing of DSBs and the frequency of crossing over, but not the frequency of gene conversion. These results suggest that Exo1 functions in the processing of DSB ends and in meiotic crossing over.
The Tumor Suppressor p53 Can Both Stimulate and Inhibit Ultraviolet Light–induced Apoptosis
Authors: McKay Bruce C. , Chen Feng , Perumalswami Chithra R. , Zhang Fenfen , Ljungman Mats
Published: July 2013

We have previously shown that the tumor suppressor p53 can play a protective role against UV-induced apoptosis in human fibroblasts. In the present study, we investigated whether the protective function of p53 expression is established before or after UV irradiation. Using a stable human cell line expressing a murine temperature-sensitive p53 in which p53 function could be tightly and reversibly regulated, we found that functional p53 stimulated the induction of apoptosis when expressed for as little as 4–12 h after UV irradiation and that this induction was not dependent on de novo protein synthesis. In contrast, expression of p53 for 12 h or more before UV irradiation reduced the extent of apoptosis even when functional p53 expression was maintained after irradiation. The protection conferred by p53 required ongoing protein synthesis and correlated with enhanced recovery of mRNA synthesis. Together, these results suggest that p53 induces distinct proapoptotic and antiapoptotic signals and that these opposing activities can be separated both temporally and by their requirement for de novo protein synthesis. These findings may have important implications for the refinement of gene therapy approaches combining p53 with pharmacological agents that target transcription or translation.
Analysis of Cortical Flow Models In Vivo
Authors: Benink Hélène A. , Mandato Craig A. , Bement William M.
Published: July 2013

Cortical flow, the directed movement of cortical F-actin and cortical organelles, is a basic cellular motility process. Microtubules are thought to somehow direct cortical flow, but whether they do so by stimulating or inhibiting contraction of the cortical actin cytoskeleton is the subject of debate. Treatment ofXenopus oocytes with phorbol 12-myristate 13-acetate (PMA) triggers cortical flow toward the animal pole of the oocyte; this flow is suppressed by microtubules. To determine how this suppression occurs and whether it can control the direction of cortical flow, oocytes were subjected to localized manipulation of either the contractile stimulus (PMA) or microtubules. Localized PMA application resulted in redirection of cortical flow toward the site of application, as judged by movement of cortical pigment granules, cortical F-actin, and cortical myosin-2A. Such redirected flow was accelerated by microtubule depolymerization, showing that the suppression of cortical flow by microtubules is independent of the direction of flow. Direct observation of cortical F-actin by time-lapse confocal analysis in combination with photobleaching showed that cortical flow is driven by contraction of the cortical F-actin network and that microtubules suppress this contraction. The oocyte germinal vesicle serves as a microtubule organizing center inXenopus oocytes; experimental displacement of the germinal vesicle toward the animal pole resulted in localized flow away from the animal pole. The results show that 1) cortical flow is directed toward areas of localized contraction of the cortical F-actin cytoskeleton; 2) microtubules suppress cortical flow by inhibiting contraction of the cortical F-actin cytoskeleton; and 3) localized, microtubule-dependent suppression of actomyosin-based contraction can control the direction of cortical flow. We discuss these findings in light of current models of cortical flow.
The Interaction Between Ran and NTF2 is Required for Cell Cycle Progression
Authors: Quimby B. Booth , Wilson Cassandra A. , Corbett Anita H.
Published: July 2013

The small GTPase Ran is required for the trafficking of macromolecules into and out of the nucleus. Ran also has been implicated in cell cycle control, specifically in mitotic spindle assembly. In interphase cells, Ran is predominately nuclear and thought to be GTP bound, but it is also present in the cytoplasm, probably in the GDP-bound state. Nuclear transport factor 2 (NTF2) has been shown to import RanGDP into the nucleus. Here, we examine the in vivo role of NTF2 in Ran import and the effect that disruption of Ran imported into the nucleus has on the cell cycle. A temperature-sensitive (ts) mutant of Saccharomyces cerevisiae NTF2 that does not bind to Ran is unable to import Ran into the nucleus at the nonpermissive temperature. Moreover, when Ran is inefficiently imported into the nucleus, cells arrest in G2in aMAD2 checkpoint-dependent manner. These findings demonstrate that NTF2 is required to transport Ran into the nucleus in vivo. Furthermore, we present data that suggest that depletion of nuclear Ran triggers a spindle-assembly checkpoint-dependent cell cycle arrest.
Differential Modulation of Cadherin-mediated Cell–Cell Adhesion by Platelet Endothelial Cell Adhesion Molecule-1 Isoforms through Activation of Extracellular Regulated Kinases
Authors: Sheibani Nader , Sorenson Christine M. , Frazier William A.
Published: July 2013

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell–cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell–cell interactions. MDCK cells lack endogenous PECAM-1 but form cell–cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Δ15 and Δ14&15, the predominant isoforms expressed in vivo. Expression of the Δ15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, α- and β-catenin expression, and sustained activation of extracellular regulated kinases. The Δ15 isoform was not concentrated at cell–cell contacts. In contrast, the Δ14&15 isoform localized to sites of cell–cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.
The Secretory Carrier Membrane Protein Family: Structure and Membrane Topology
Authors: Hubbard Charles , Singleton David , Rauch Michelle , Jayasinghe Sajith , Cafiso David , Castle David
Published: July 2013

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain–binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32–38 kDa except mammalian SCAMP4, which is ∼25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an ∼20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is α-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.