A Rab2 Mutant with Impaired GTPase Activity Stimulates Vesicle Formation from Pre-Golgi Intermediates

Rab2 immunolocalizes to pre-Golgi intermediates (vesicular-tubular clusters [VTCs]) that are the first site of segregation of anterograde- and retrograde-transported proteins and a major peripheral site for COPI recruitment. Our previous work showed that Rab2 Q65L (equivalent to Ras Q61L) inhibited endoplasmic reticulum (ER)-to-Golgi transport in vivo. In this study, the biochemical properties of Rab2 Q65L were analyzed. The mutant protein binds GDP and GTP and has a low GTP hydrolysis rate that suggests that Rab2 Q65L is predominantly in the GTP-bound–activated form. The purified protein arrests vesicular stomatitis virus glycoprotein transport from VTCs in an assay that reconstitutes ER-to-Golgi traffic. A quantitative binding assay was used to measure membrane binding of β-COP when incubated with the mutant. Unlike Rab2 that stimulates recruitment, Rab2 Q65L showed a dose-dependent decrease in membrane-associated β-COP when incubated with rapidly sedimenting membranes (ER, pre-Golgi, and Golgi). The mutant protein does not interfere with β-COP binding but stimulates the release of slowly sedimenting vesicles containing Rab2, β-COP, and p53/gp58 but lacking anterograde grade-directed cargo. To complement the biochemical results, we observed in a morphological assay that Rab2 Q65L caused vesiculation of VTCs that accumulated at 15°C. These data suggest that the Rab2 protein plays a role in the low-temperature–sensitive step that regulates membrane flow from VTCs to the Golgi complex and back to the ER.

Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins’ dominant negative effects in vivo.

An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.

A Specific Point Mutant at Position 1 of the Influenza Hemagglutinin Fusion Peptide Displays a Hemifusion Phenotype

We showed previously that substitution of the first residue of the influenza hemagglutinin (HA) fusion peptide Gly1 with Glu abolishes fusion activity. In the present study we asked whether this striking phenotype was due to the charge or side-chain volume of the substituted Glu. To do this we generated and characterized six mutants with substitutions at position 1: Gly1 to Ala, Ser, Val, Glu, Gln, or Lys. We found the following. All mutants were expressed at the cell surface, could be cleaved from the precursor (HA0) to the fusion permissive form (HA1-S-S-HA2), bound antibodies against the major antigenic site, bound red blood cells, and changed conformation at low pH. Only Gly, Ala, and Ser supported lipid mixing during fusion with red blood cells. Only Gly and Ala supported content mixing. Ser HA, therefore, displayed a hemifusion phenotype. The hemifusion phenotype of Ser HA was confirmed by electrophysiological studies. Our findings indicate that the first residue of the HA fusion peptide must be small (e.g., Gly, Ala, or Ser) to promote lipid mixing and must be small and apolar (e.g., Gly or Ala) to support both lipid and content mixing. The finding that Val HA displays no fusion activity underscores the idea that hydrophobicity is not the sole factor dictating fusion peptide function. The surprising finding that Ser HA displays hemifusion suggests that the HA ectodomain functions not only in the first stage of fusion, lipid mixing, but also, either directly or indirectly, in the second stage of fusion, content mixing.

A p90rsk Mutant Constitutively Interacting with MAP Kinase Uncouples MAP Kinase from p34cdc2/Cyclin B Activation in Xenopus Oocytes

The efficient activation of p90rsk by MAP kinase requires their interaction through a docking site located at the C-terminal end of p90rsk. The MAP kinase p42mpk1 can associate with p90rsk in G2-arrested but not in mature Xenopus oocytes. In contrast, an N-terminally truncated p90rsk mutant named D2 constitutively interacts with p42mpk1. In this report we show that expression of D2 inhibits Xenopus oocyte maturation. The inhibition requires the p42mpk1 docking site. D2 expression uncouples the activation of p42mpk1 and p34cdc2/cyclin B in response to progesterone but does not prevent signaling through p90rsk. Instead, D2 interferes with a p42mpk1-triggered pathway, which regulates the phosphorylation and activation of Plx1, a potential activator of the Cdc25 phosphatase. This new pathway that links the activation of p42mpk1 and Plx1 during oocyte maturation is independent of p34cdc2/cyclin B activity but requires protein synthesis. Using D2, we also provide evidence that the sustained activation of p42mpk1 can trigger nuclear migration in oocytes. Our results indicate that D2 is a useful tool to study MAP kinase function(s) during oocyte maturation. Truncated substrates such as D2, which constitutively interact with MAP kinases, may also be helpful to study signal transduction by MAP kinases in other cellular processes.

Derepressed Hyphal Growth and Reduced Virulence in a VH1 Family-related Protein Phosphatase Mutant of the Human Pathogen Candida albicans

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.

A Mutant of Arp2p Causes Partial Disassembly of the Arp2/3 Complex and Loss of Cortical Actin Function in Fission Yeast

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2+ gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

A highly digestible sorghum mutant cultivar exhibits a unique folded structure of endosperm protein bodies

The endosperm of a sorghum mutant cultivar, with high in vitro uncooked and cooked protein digestibilities, was examined by transmission electron microscopy and α-, β-, and γ-kafirins (storage proteins) were localized within its protein bodies. Transmission electron microscopy micrographs revealed that these protein bodies had a unique microstructure related to high protein digestibility. They were irregular in shape and had numerous invaginations, often reaching to the central area of the protein body. Protein bodies from normal cultivars, such as P721N studied here, with much lower uncooked and cooked digestibilities are spherical and contain no invaginations. Immunocytochemistry results showed that the relative location of α- and β-kafirins within the protein bodies of the highly digestible genotype were similar to the normal cultivar, P721N. γ-Kafirin, however, was concentrated in dark-staining regions at the base of the folds instead of at the protein body periphery, as is typical of normal cultivars. The resulting easy accessibility of digestive enzymes to α-kafirin, the major storage protein, in addition to the increased surface area of the protein bodies of the highly digestible cultivar appear to account for its high in vitro protein digestibility.

Deficits in memory and motor performance in synaptotagmin IV mutant mice

Synaptotagmin (Syt) IV is a synaptic vesicle protein. Syt IV expression is induced in the rat hippocampus after systemic kainic acid treatment. To examine the functional role of this protein in vivo, we derived Syt IV null [Syt IV(−/−)] mutant mice. Studies with the rotorod revealed that the Syt IV mutants have impaired motor coordination, a result consistent with constitutive Syt IV expression in the cerebellum. Because Syt IV is thought to modulate synaptic function, we also have examined Syt IV mutant mice in learning and memory tests. Our studies show that the Syt IV mutation disrupts contextual fear conditioning, a learning task sensitive to hippocampal and amygdala lesions. In contrast, cued fear conditioning is normal in the Syt IV mutants, suggesting that this mutation did not disrupt amygdala function. Conditioned taste aversion, which also depends on the amygdala, is normal in the Syt IV mutants. Consistent with the idea that the Syt IV mutation preferentially affects hippocampal function, Syt IV mutant mice also display impaired social transmission of food preference. These studies demonstrate that Syt IV is critical for brain function and suggest that the Syt IV mutation affects hippocampal-dependent learning and memory, as well as motor coordination.

Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution

Mutant alleles at the dilute unconventional myosin heavy chain locus cause diluted coat color, opisthotonic seizures, and death. The dilute coat color phenotype is caused by irregular clumping of pigment in the hair, but amounts of melanin are unchanged from wild-type controls. The melanocyte phenotype has been described as adendritic, since hair bulb and Harderian gland melanocytes appear to be rounded in tissue sections. These observations do not exclude the possibility that the processes lack pigment, since the melanocyte shape was judged by the distribution of melanin. We have tested this hypothesis by culturing primary melanocytes from dilute mutant and wild-type mice. The mutant melanocytes do not lack processes; instead, they exhibit a concentrated perinuclear distribution of melanosomes, while wild-type melanocytes have a very uniform cytoplasmic distribution of melanosomes. Electron micrographs show no detectable differences in melanosome morphology or maturation between dilute and wild-type melanocytes. Immunofluorescence experiments indicate that the dilute protein is concentrated in regions of the cytoplasm that contain melanosomes. These experiments show that the dilute myosin is necessary for the localization of melanosomes, either by active transport or tethering.

Length suppression in histone messenger RNA 3′-end maturation: Processing defects of insertion mutant premessenger RNAs can be compensated by insertions into the U7 small nuclear RNA

Efficient 3′-end processing of cell cycle-regulated mammalian histone premessenger RNAs (pre-mRNAs) requires an upstream stem–loop and a histone downstream element (HDE) that base pairs with the U7 small ribonuclearprotein. Insertions between these elements have two effects: the site of cleavage moves in concert with the HDE and processing efficiency declines. We used Xenopus oocytes to ask whether compensatory length insertions in the human U7 RNA could restore the fidelity and efficiency of processing of mouse histone insertion pre-mRNAs. An insertion of 5 nt into U7 RNA that extends its complementary to the HDE compensated for both defects in processing of a 5-nt insertion substrate; a noncomplementary insertion into U7 did not. Yet, the noncomplementary insertion mutant U7 was shown to be active on insertion substrates further mutated to allow base pairing. Our results suggest that the histone pre-mRNA becomes rigidified upstream of its HDE, allowing the bound U7 small ribonucleoprotein to measure from the HDE to the cleavage site. Such a mechanism may be common to other RNA measuring systems. To our knowledge, this is the first demonstration of length suppression in an RNA processing system.

Characterization of a replication-incompetent adenovirus type 5 mutant deleted for the preterminal protein gene

An adenovirus type 5 mutant deleted for the preterminal protein (pTP) gene was constructed using cell lines that express pTP. The pTP deletion mutant virus is incapable of replicating in the absence of complementation and does not express detectable levels of viral mRNAs that are expressed only after the onset of replication. Accumulation of early-region mRNAs, including that for E1A, exhibits a lag relative to that observed from the wild-type virus. However, E1A mRNA accumulation attains a steady-state level similar to the level of expression during the early phase of infection with the wild-type virus. In 293-pTP cells (human embryonic kidney cells that express pTP in addition to high levels of adenovirus E1A and E1B proteins), the pTP deletion mutant virus replicates efficiently and yields infectious titers within 5-fold of that of the wild-type virus. The deletion of 1.2 kb of pTP-encoding sequence increases the size of foreign DNA that can be introduced into the virus and, with an absolute block to replication, makes this virus an important tool for gene therapy.

Recombinant immunotoxins specific for a mutant epidermal growth factor receptor: Targeting with a single chain antibody variable domain isolated by phage display

EGFRvIII is a mutant epidermal growth factor receptor found in glioblastoma, and in carcinoma of the breast, ovary, and lung. The mutant receptor has a deletion in its extracellular domain that results in the formation of a new, tumor-specific extracellular sequence. Mice were immunized with a synthetic peptide corresponding to this sequence and purified EGFRvIII. A single chain antibody variable domain (scFv) phage display library of 8 × 106 members was made from the spleen of one immunized mouse. A scFv specific for EGFRvIII was isolated from this library by panning with successively decreasing amounts of synthetic peptide. This was used to make an immunotoxin by fusing the scFv DNA sequence to sequences coding for domains II and III of Pseudomonas exotoxin A. Purified immunotoxin had a Kd of 22 nM for peptide and a Kd of 11 nM for cell-surface EGFRvIII. The immunotoxin was very cytotoxic to cells expressing EGFRvIII, with an IC50 of 1 ng/ml (16 pM) on mouse fibroblasts transfected with EGFRvIII and an IC50 of 7–10 ng/ml (110–160 pM) on transfected glioblastoma cells. There was no cytotoxic activity at 1000 ng/ml on the untransfected parent glioblastoma cell line. The immunotoxin was completely stable upon incubation at 37°C for 24 h in human serum. The combination of good affinity, cytotoxicity and stability make this immunotoxin a candidate for further preclinical evaluation.

Assessment of normal and mutant human presenilin function in Caenorhabditis elegans

We provide evidence that normal human presenilins can substitute for Caenorhabditis elegans SEL-12 protein in functional assays in vivo. In addition, six familial Alzheimer disease-linked mutant human presenilins were tested and found to have reduced ability to rescue the sel-12 mutant phenotype, suggesting that they have lower than normal presenilin activity. A human presenilin 1 deletion variant that fails to be proteolytically processed and a mutant SEL-12 protein that lacks the C terminus display considerable activity in this assay, suggesting that neither presenilin proteolysis nor the C terminus is absolutely required for normal presenilin function. We also show that sel-12 is expressed in most neural and nonneural cell types in all developmental stages. The reduced activity of mutant presenilins and as yet unknown gain-of-function properties may be a contributing factor in the development of Alzheimer disease.

Kinetic and structural analysis of mutant CD4 receptors that are defective in HIV gp120 binding

The T-cell antigen coreceptor CD4 also serves as the receptor for the envelope glycoprotein gp120 of HIV. Extensive mutational analysis of CD4 has implicated residues from a portion of the extracellular amino-terminal domain (D1) in gp120 binding. However, none of these proteins has been fully characterized biophysically, and thus the precise effects on molecular structure and binding interactions are unknown. In the present study, we produced soluble versions of three mutant CD4 molecules (F43V, G47S, and A55F) and characterized their structural properties, thermostability, and ability to bind gp120. Crystallographic and thermodynamic analysis showed minimal structural alterations in the F43V and G47S mutant proteins, which have solvent-exposed mutant side chains. In contrast, some degree of disorder appears to exist in the folded state of A55F, as a result of mutating a buried side chain. Real time kinetic measurements of the interaction of the mutant proteins with gp120 showed affinity decreases of 5-fold for G47S, 50-fold for A55F, and 200-fold for F43V. Although both rate constants for the binding reaction were affected by these mutations, the loss in affinity was mainly due to a decrease in on rates, with less drastic changes occurring in the off rates. These observations suggest the involvement of conformational adaptation in the CD4–gp120 interaction. Together, the structural and kinetic data confirm that F43V is a critical residue in gp120 recognition site, which may also include main chain interactions at residue Gly-47.