Regulation of Sos activity by intramolecular interactions

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.     

Two separate functions are encoded by the carboxyl-terminal domains of the yeast cyclase-associated protein and its mammalian homologs. Dimerization and actin binding

The yeast adenylyl cyclase-associated protein, CAP, was identified as a component of the RAS-activated cyclase complex. CAP consists of two functional domains separated by a proline-rich region. One domain, which localizes to the amino terminus, mediates RAS signaling through adenylyl cyclase, while a domain at the carboxyl terminus is involved in the regulation of cell growth and morphogenesis. Recently, the carboxyl terminus of yeast CAP was shown to sequester actin, but whether this function has been conserved, and is the sole function of this domain, is unclear. Here, we demonstrate that the carboxyl-terminal domains of CAP and CAP homologs have two separate functions. We show that carboxyl-terminals of both yeast CAP and a mammalian CAP homolog, MCH1, bind to actin. We also show that this domain contains a signal for dimerization, allowing both CAP and MCH1 to form homodimers and heterodimers. The properties of actin binding and dimerization are mediated by separate regions on the carboxyl terminus; the last 27 amino acids of CAP being critical for actin binding. Finally, we present evidence that links a segment of the proline-rich region of CAP to its localization in yeast. Together, these results suggest that all three domains of CAP proteins are functional.     

Analysis of the thrombin inhibitor DuP 714 by an enzyme-linked immunosorbent assay

Resident proteins of the exocytic pathway contain at least two types of information in their primary sequence for determining their subcellular location. The first type of information is found at the carboxyl terminus of soluble proteins of the endoplasmic reticulum (ER) and in the cytoplasmic domain of some ER and Golgi membrane proteins. It acts as a retrieval signal, returning proteins that have left the compartment in which they reside. The second type of information has been found in the membrane-spanning domain of several ER and Golgi proteins and, though the mechanism by which it operates is still unclear, it acts as a retention signal, keeping the protein at a particular location within the organelle. The presence of both a retrieval signal and a retention signal in a trans-Golgi network resident protein suggests that more than one mechanism operates to ensure correct localization of resident proteins along the exocytic pathway.     

The role in cell binding of a beta-bend within the triple helical region in collagen alpha 1 (I) chain: structural and biological evidence for conformational tautomerism on fiber surface

Dystrophin is a plasma membrane-associated cytoskeletal protein of the spectrin superfamily. The dystrophin cytoskeleton has been first characterized in muscle. Muscular 427 kDa dystrophin binds to subplasmalemmal actin filaments via its amino-terminal domain. The carboxy-terminus of dystrophin binds to a plasma membrane anchor, beta-dystroglycan, which is associated on the external side with the extracellular matrix receptor, alpha-dystroglycan, that binds to the basal lamina proteins laminin-1, laminin-2, and agrin. In the muscle, the dystroglycan complex is associated with the sarcoglycan complex that consists of several glycosylated, integral membrane proteins. The absence or functional deficiency of the dystrophin cytoskeleton is the cause of several types of muscular dystrophies including the lethal Duchenne muscular dystrophy (DMD), one of the most severe and most common genetic disorders of man. The dystrophin complex is believed to stabilize the plasma membrane during cycles of contraction and relaxation. Muscular dystrophin and several types of dystrophin variants are also present in extramuscular tissues, e.g. in distinct regions of the central nervous systems including the retina. Absence of dystrophin from these sites is believed to be responsible for some extramuscular symptoms of DMD, e.g. mental retardation and disturbances in retinal electrophysiology (reduced b-wave in electroretinograms). The reduced b-wave in electroretinograms indicated a disturbance of neurotransmission between photoreceptors and ON-bipolar cells. At least two different dystrophin variants are present in photoreceptor synaptic complexes. One of these dystrophins (Dp260) is virtually exclusively expressed in the retina. In the neuroretina, dystrophin is found in significant amounts in the invaginated photoreceptor synaptic complexes. At this location dystrophin colocalizes with dystroglycan. Agrin, an extracellular ligand of alpha-dystroglycan, is also present at this location whereas the proteins of the sarcoglycan complex appear to be absent in photoreceptor synaptic complexes. Dystrophin and dystroglycan are located distal from the ribbon-containing active synaptic zones where both proteins are restricted to the photoreceptor plasma membrane bordering on the lateral sides of the synaptic invagination. In addition, some neuronal profiles of the postsynaptic complex also contain dystrophin and beta-dystroglycan. These profiles appear to belong at least in part to projections of the photoreceptor terminals into the postsynaptic dendritic complex. In view of the abnormal neurotransmission between photoreceptors and ON-bipolar cells in DMD patients the dystrophin/beta-dystroglycan-containing projections of photoreceptor presynaptic terminals into the postsynaptic dendritic plexus might somehow modify the ON-bipolar pathway. Another retinal site associated with dystrophin/beta-dystropglycan is the plasma membrane of Müller cells where dystrophin/beta-dystroglycan appear to be present at particular high concentrations. At this location the dystrophin/dystroglycan complex may play a role in the attachment of the retina to the vitreous, and, under pathological conditions, in traction-induced retinal detachment.     

The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane

Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Deltaste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues.     

Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.     

Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (approximately 5 microM) than those in the cytoplasm (approximately 100 nM); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (approximately 420 nM) were twice those measured by connexin-32 chimera (approximately 200 nM); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (approximately 130 nM). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.     

Role of the TonB amino terminus in energy transduction between membranes

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.     

Identification of a novel ankyrin isoform (AnkG190) in kidney and lung that associates with the plasma membrane and binds alpha-Na, K-ATPase

Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins. Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. We have cloned and characterized a novel 190-kDa member of this family from a rat kidney cDNA library, which we term AnkG190 based on the predicted size and homology with the larger neuronal AnkG isoform. AnkG190 displays a unique 31-residue amino terminus, a repeats domain consisting of 24 repetitive 33-residue motifs, a spectrin binding domain, and a truncated regulatory domain. Probes derived from the unique amino terminus hybridize to an 8-kilobase message exclusively in kidney and lung and specifically to the kidney outer medullary collecting ducts by in situ hybridization. Transfections of Madin-Darby canine kidney and COS-7 epithelial cell lines with a full-length AnkG190 construct result in (a) expression at the lateral plasma membrane, (b) functional assembly with the cytoskeleton, and (c) interaction with at least one membrane protein, the Na,K-ATPase. Two independent Na,K-ATPase binding domains on AnkG190 are demonstrated as follows: one within the distal 12 ankyrin repeats, and a second site within the spectrin binding domain. Thus, ankyrins may interact with integral membrane proteins in a pleiotropic manner that may involve complex tertiary structural determinants.     

Dystrophin, utrophin and beta-dystroglycan expression in skeletal muscle from patients with Becker muscular dystrophy

The precise localization and semiquantitative correlation of dystrophin, utrophin and beta-dystroglycan expression on the sarcolemma of skeletal muscle cells obtained from patients with Becker muscular dystrophy (BMD) was studied using three types of double immunofluorescence. Staining intensity was measured using a confocal laser microscope. Each of these proteins was identified at the same locus on the sarcolemma. The staining intensities of dystrophin and utrophin were approximately reciprocal at sarcolemmal sites where dystrophin expression was obviously observed. The staining intensity of beta-dystroglycan was strong in areas where dystrophin staining was also strong and utrophin expression was weak. Quantitative analysis revealed that the staining intensity of beta-dystroglycan minus that of dystrophin approximated the staining intensity of utrophin, indicating that the sum of dystrophin and utrophin expression corresponds to that of beta-dystroglycan. These results suggest that utrophin may compensate for dystrophin deficiency found in BMD by binding to beta-dystroglycan.     

Chp, a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton

The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.     

Transforming growth factor beta1 induces nuclear export of inhibitory Smad7

Transforming growth factor beta (TGF-beta) signals from membrane to nucleus through serine/threonine kinase receptors and their downstream effector molecules, termed Smad proteins. Recently, Smad6 and Smad7 were identified, which antagonize TGF-beta family signaling by preventing the activation of signal-transducing Smad complexes. Here we report that Smad7, but not Smad6, inhibits TGF-beta1-induced growth inhibition and the expression of immediate early response genes, including Smad7. Interestingly, in the absence of ligand, Smad7 was found to be predominantly localized in the nucleus, whereas Smad7 accumulated in the cytoplasm upon TGF-beta receptor activation. The latter is in accordance with the physical association of Smad7 with the ligand-activated TGF-beta receptor complex in the cell membrane. Whereas the ectopically expressed C-terminal domain of Smad7 was also exported from the nucleus to the cytoplasm upon TGF-beta challenge, a Smad7 mutant with a small deletion at the C terminus or only the N-terminal domain of Smad7 was localized mainly in the cytoplasm in the absence or presence of ligand. This suggests that an intact Mad homology 2 domain is important for nuclear localization of Smad7. The nuclear localization of Smad7 suggests a functional role distinct from its antagonistic effect in receptor-mediated Smad activation.     

Septal localization of FtsQ, an essential cell division protein in Escherichia coli

Septation in Escherichia coli requires several gene products. One of these, FtsQ, is a simple bitopic membrane protein with a short cytoplasmic N terminus, a membrane-spanning segment, and a periplasmic domain. We have constructed a merodiploid strain that expresses both FtsQ and the fusion protein green fluorescent protein (GFP)-FtsQ from single-copy chromosomal genes. The gfp-ftsQ gene complements a null mutation in ftsQ. Fluorescence microscopy revealed that GFP-FtsQ localizes to the division site. Replacing the cytoplasmic and transmembrane domains of FtsQ with alternative membrane anchors did not prevent the localization of the GFP fusion protein, while replacing the periplasmic domain did, suggesting that the periplasmic domain is necessary and sufficient for septal targeting. GFP-FtsQ localization to the septum depended on the cell division proteins FtsZ and FtsA, which are cytoplasmic, but not on FtsL and FtsI, which are bitopic membrane proteins with comparatively large periplasmic domains. In addition, the septal localization of ZipA apparently did not require functional FtsQ. Our results indicate that FtsQ is an intermediate recruit to the division site.     

Structure-function analysis of the Bcl-2 oncoprotein. Addition of a heterologous transmembrane domain to portions of the Bcl-2 beta protein restores function as a regulator of cell survival

The bcl-2 gene can potentially encode 26- and 22-kDa proteins that differ only in their carboxyl tails because of an alternative splicing mechanism. The larger of these proteins contains a hydrophobic transmembrane domain within its carboxyl terminus, resides (at least in part) in mitochondrial membranes and has been shown to prolong cell survival by blocking programmed cell death (also termed "apoptosis"). To explore the function of the shorter 22-kDa Bcl-2 protein that lacks a transmembrane domain, DNAs encoding p26-Bcl-2-alpha or p22-Bcl-2-beta were expressed in an interleukin-3 (IL-3)-dependent hematopoietic cell line 32D. In contrast to p26-Bcl-2 alpha that markedly prolonged cell survival, p22-Bcl-2-beta did not extend the survival of 32D cells when cultured in the absence of IL-3. Expression in 32D cells of a chimeric DNA that fused portions of the open reading frame common to Bcl-2-alpha and Bcl-2-beta (amino-acids 1-195) with sequences encoding the transmembrane and cytosolic domains of the IL-2 receptor-alpha protein resulted in production of a Bcl-2/IL-2R fusion protein that was capable of prolonging 32D cell survival in the setting of IL-3 withdrawal. Based on fractionation of cells to produce crude heavy membrane, light membrane, nuclei, and cytosolic preparations, much of the p22-Bcl-2-beta protein appeared to reside in the cytosol, whereas Bcl-2-alpha and the Bcl-2/IL-2R chimeric proteins were found exclusively in fractions that also contained the inner mitochondrial membrane protein F1-beta-ATPase. Taken together, these findings demonstrate the importance of membrane association for the function and intracellular targeting of the apoptosis-blocking Bcl-2 protein. Furthermore, despite the strong evolutionary conservation of the carboxyl regions of Bcl-2-alpha proteins observed previously for mammalian and avian species, these data suggest that a heterologous transmembrane domain can be substituted without loss of function.     

Ca2+ differentially regulates conventional protein kinase Cs' membrane interaction and activation

The regulation of conventional protein kinase Cs by Ca2+ was examined by determining how this cation affects the enzyme's 1) membrane binding and catalytic function and 2) conformation. In the first part, we show that significantly lower concentrations of Ca2+ are required to effect half-maximal membrane binding than to half-maximally activate the enzyme. The disparity between binding and activation kinetics is most striking for protein kinase C betaII, where the concentration of Ca2+ promoting half-maximal membrane binding is approximately 40-fold higher than the apparent Km for Ca2+ for activation. In addition, the Ca2+ requirement for activation of protein kinase C betaII is an order of magnitude greater than that for the alternatively spliced protein kinase C betaI; these isozymes differ only in 50 amino acids at the carboxyl terminus, revealing that residues in the carboxyl terminus influence the enzyme's Ca2+ regulation. In the second part, we use proteases as conformational probes to show that Ca2+dependent membrane binding and Ca2+-dependent activation involve two distinct sets of structural changes in protein kinase C betaII. Three separate domains spanning the entire protein participate in these conformational changes, suggesting significant interdomain interactions. A highly localized hinge motion between the regulatory and catalytic halves of the protein accompanies membrane binding; release of the carboxyl terminus accompanies the low affinity membrane binding mediated by concentrations of Ca2+ too low to promote catalysis; and exposure of the amino-terminal pseudosubstrate and masking of the carboxyl terminus accompany catalysis. In summary, these data reveal that structural determinants unique to each isozyme of protein kinase C dictate the enzyme's Ca2+-dependent affinity for acidic membranes and show that, surprisingly, some of these determinants are in the carboxyl terminus of the enzyme, distal from the Ca2+-binding site in the amino-terminal regulatory domain.     

A dystrophin missense mutation showing persistence of dystrophin and dystrophin-associated proteins yet a severe phenotype

A muscle biopsy from an X-linked muscular dystrophy pedigree showed normal dystrophin and dystrophin-associated proteins. Linkage to multiple markers within the dystrophin gene (LOD=2.7, theta=0) indicated a primary dystrophinopathy. Sequencing of the entire dystrophin RNA revealed a single missense mutation (D3335H) in the unique carboxyl-terminal domain. This is the first report showing that a relatively severe dystrophinopathy can occur despite the correct localization of dystrophin and dystrophin-associated proteins.     

Rab2 is essential for the maturation of pre-Golgi intermediates

The small GTPase Rab2 is a resident of pre-Golgi intermediates and required for protein transport from the endoplasmic reticulum (ER) to the Golgi complex (Tisdale, E. J., Bourne, J. R., Khosravi-Far, R. , Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761). The Rab2 protein, like all small GTPases, contains conserved GTP-binding domains as well as hypervariable carboxyl-terminal and amino-terminal domains. While the role of the carboxyl terminus in specific membrane localization is well recognized, the potential role of the variable NH2 terminus remains to be clarified. To determine whether the NH2 terminus of Rab2 was required for its activity in vivo, a trans dominant mutant of Rab2 that inhibits ER to Golgi transport was progressively truncated and analyzed for its effect on vesicular stomatitis virus glycoprotein transport in a vaccinia-based transient expression system. Deletion of the first 14 amino-terminal residues resulted in the loss of the inhibitory properties of the mutant without affecting its post-translational processing or membrane association. To assess the potential role of the NH2 terminus in Rab2 function, a peptide corresponding to the first 13 amino acids following the initiator methionine was introduced into an in vitro assay that efficiently reconstitutes transport of vesicular stomatitis virus glycoprotein from the ER to the Golgi stack. This peptide was a potent inhibitor of transport. Biochemical and morphological studies revealed that the peptide strongly interfered with assembly of pre-Golgi intermediates which mediate segregation of anterograde and retrograde transported proteins en route to the Golgi. The combined results suggest that the NH2 terminus of Rab2 is required for its function and for direct interaction with components of the transport machinery involved in the maturation of pre-Golgi intermediates.     

In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei

Duchenne muscular dystrophy is frequently associated with a non-progressive cognitive deficit attributed to the absence of 427,000 mol. wt brain dystrophin, or to altered expression of other C-terminal products of this protein, Dp71 and/or Dp140. To further explore the role of these membrane cytoskeleton-associated proteins in brain function, we studied spatial learning and ex vivo synaptic plasticity in the mdx mouse, which lacks 427,000 mol. wt dystrophin, and in the mdx3cv mutant, which shows a dramatically reduced expression of all the dystrophin gene products known so far. We show that reference and working memories are largely unimpaired in the two mutant mice performing a spatial discrimination task in a radial maze. However, mdx3cv mice showed enhanced emotional reactivity and developed different strategies in learning the task, as compared to control mice. We also showed that both mutants display apparently normal levels of long-term potentiation and paired-pulse facilitation in the CA1 field of the hippocampus. On the other hand, an increased post-tetanic potentiation was shown by mdx, but not mdx3cv mice, which might be linked to calcium-regulatory defects. Otherwise, immunoblot analyses suggested an increased expression of a 400,000 mol. wt protein in brain extracts from both mdx and mdx3cv mice, but not in those from control mice. This protein might correspond to the dystrophin-homologue utrophin. The present results suggest that altered expression of dystrophin or C-terminal dystrophin proteins in brain did not markedly affect hippocampus-dependent spatial learning and CA1 hippocampal long-term potentiation in mdx and mdx3cv mice. The role of these membrane cytoskeleton-associated proteins in normal brain function and pathology remains to be elucidated. Furthermore, the possibility that redundant mechanisms could partially compensate for dystrophins' deficiency in the mdx and mdx3cv models should be further considered.     

Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p

GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.