High-affinity binding of Escherichia coli SecB to the signal sequence region of a presecretory protein.

The Escherichia coli cytosolic homotetrameric protein SecB is known to be involved in protein export across the plasma membrane. A currently prevalent view holds that SecB functions exclusively as a chaperone interacting nonspecifically with unfolded proteins, not necessarily exported proteins, whereas a contrary view holds that SecB functions primarily as a specific signal-recognition factor--i.e., in binding to the signal sequence region of exported proteins. To experimentally resolve these differences we assayed for binding between chemically pure SecB and chemically pure precursor (p) form (containing a signal sequence) and mature (m) form (lacking a signal sequence) of a model secretory protein (maltose binding protein, MBP) that was C-terminally truncated. Because of the C-terminal truncation, neither p nor m was able to fold. We found that SecB bound with 100-fold higher affinity to p (Kd 0.8 nM) than it bound to m (Kd 80 nM). As the presence of the signal sequence in p is the only feature that distinguished p from m, these data strongly suggest that the high-affinity binding of SecB is to the signal sequence region and not the mature region of p. Consistent with this conclusion, we found that a wild-type signal peptide, but not an export-incompetent mutant signal peptide of another exported protein (LamB), competed for binding to p. Moreover, the high-affinity binding of SecB to p was resistant to 1 M salt, whereas the low-affinity binding of SecB to m was not. These qualitative differences suggested that SecB binding to m was primarily by electrostatic interactions, whereas SecB binding to p was primarily via hydrophobic interactions, presumably with the hydrophobic core of the signal sequence. Taken together our data strongly support the notion that SecB is primarily a specific signal-recognition factor.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain.

12(S)-hydroxyeicosatetraenoic acid and 13(S)-hydroxyoctadecadienoic acid regulation of protein kinase C-alpha in melanoma cells: role of receptor-mediated hydrolysis of inositol phospholipids.

Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.

Apolipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals.

Apolipoprotein (apo-) B mRNA editing is the deamination of cytidine that creates a new termination codon and produces a truncated version of apo-B (apo-B48). The cytidine deaminase catalytic subunit [apo-B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1)] of the multiprotein editing complex has been identified. We generated transgenic rabbits and mice expressing rabbit APOBEC-1 in their livers to determine whether hepatic expression would lower low density lipoprotein cholesterol concentrations. The apo-B mRNA from the livers of the transgenic mice and rabbit was extensively edited, and the transgenic animals had reduced concentrations of apo-B100 and low density lipoproteins compared with control animals. Unexpectedly, all of the transgenic mice and a transgenic rabbit had liver dysplasia, and many transgenic mice developed hepatocellular carcinomas. Many of the mouse livers were hyperplastic and filled with lipid. Other hepatic mRNAs with sequence motifs similar to apo-B mRNA were examined for this type of editing (i.e., cytidine deamination). One of these, tyrosine kinase, was edited in livers of transgenic mice but not of controls. This result demonstrates that other mRNAs can be edited by the overexpressed editing enzyme and suggests that aberrant editing of hepatic mRNAs involved in cell growth and regulation is the cause of the tumorigenesis. Finally, these findings compromise the potential use of APOBEC-1 for gene therapy to lower plasma levels of low density lipoproteins.

Role of glutathione in the export of compounds from cells by the multidrug-resistance-associated protein.

Multidrug-resistance-associated protein (MRP) is a plasma membrane glycoprotein that can confer multidrug resistance (MDR) by lowering intracellular drug concentration. Here we demonstrate that depletion of intracellular glutathione by DL-buthionine (S,R)-sulfoximine results in a complete reversal of resistance to doxorubicin, daunorubicin, vincristine, and VP-16 in lung carcinoma cells transfected with a MRP cDNA expression vector. Glutathione depletion had less effect on MDR in cells transfected with MDR1 cDNA encoding P-glycoprotein and did not increase the passive uptake of daunorubicin by cells, indicating that the decrease of MRP-mediated MDR was not due to nonspecific membrane damage. Glutathione depletion resulted in a decreased efflux of daunorubicin from MRP-transfected cells, but not from MDR1-transfected cells, suggesting that glutathione is specifically required for the export of drugs from cells by MRP. We also show that MRP increases the export of glutathione from the cell and this increased export is further elevated in the presence of arsenite. Our results support the hypothesis that MRP functions as a glutathione S-conjugate carrier.

Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin.

Pro-phenol oxidase [pro-PO; zymogen of phenol oxidase (monophenol, L-dopa:oxygen oxidoreductase, EC 1.14.18.1)] is present in the hemolymph plasma of the silkworm Bombyx mori. Pro-PO is a heterodimeric protein synthesized by hemocytes. A specific serine proteinase activates both subunits through a limited proteolysis. The amino acid sequences of both subunits were deduced from their respective cDNAs; amino acid sequence homology between the subunits was 51%. The deduced amino acid sequences revealed domains highly homologous to the copper-binding site sequences (copper-binding sites A and B) of arthropod hemocyanins. The overall sequence homology between silkworm pro-PO and arthropod hemocyanins ranged from 29 to 39%. Phenol oxidases from prokaryotes, fungi, and vertebrates have sequences homologous to only the copper-binding site B of arthropod hemocyanins. Thus, silkworm pro-PO DNA described here appears distinctive and more closely related to arthropod hemocyanins. The pro-PO-activating serine proteinase was shown to hydrolyze peptide bonds at the carboxyl side of arginine in the sequence-Asn-49-Arg-50-Phe-51-Gly-52- of both subunits. Amino groups of N termini of both subunits were indicated to be N-acetylated. The cDNAs of both pro-PO subunits lacked signal peptide sequences. This result supports our contention that mature pro-PO accumulates in the cytoplasm of hemocytes and is released by cell rupture, as for arthropod hemocyanins.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Pax5 (BSAP) regulates the murine immunoglobulin 3' alpha enhancer by suppressing binding of NF-alpha P, a protein that controls heavy chain transcription.

The Pax5 transcription factor BSAP (B-cell-specific activator protein) is known to bind to and repress the activity of the immunoglobulin heavy chain 3' alpha enhancer. We have detected an element--designated alpha P--that lies approximately 50 bp downstream of the BSAP binding site 1 and is required for maximal enhancer activity. In vitro binding experiments suggest that the 40-kDa protein that binds to this element (NF-alpha P) is a member of the Ets family present in both B-cell and plasma-cell nuclei. However, in vivo footprint analysis suggests that the alpha P site is occupied only in plasma cells, whereas the BSAP site is occupied in B cells but not in plasma cells. When Pax5 binding to the enhancer in B cells was blocked in vivo by transfection with a triple-helix-forming oligonucleotide an alpha P footprint appeared and endogenous immunoglobulin heavy chain transcripts increased. The triple-helix-forming oligonucleotide also increased enhancer activity of a transfected construct in B cells, but only when the alpha P site was intact. Pax5 thus regulates the 3' alpha enhancer and immunoglobulin gene transcription by blocking activation by NF-alpha P.

Oligogalacturonide defense signals in plants: large fragments interact with the plasma membrane in vitro.

Oligogalacturonides are plant cell wall-derived regulatory molecules which stimulate defense gene expression during pathogenesis. In vitro, these compounds enhance the phosphorylation of an approximately 34-kDa protein (pp34) in purified plasma membranes from potato and tomato leaves. We now show that polygalacturonate-enhanced phosphorylation of pp34 occurs in plasma membranes purified from tomato roots, hypocotyls, and stems and from undifferentiated potato cells. Furthermore, a similar phosphorylation is detected in leaf plasma membranes from soybean, a plant distantly related to tomato. Purified oligogalacturonides 13 to at least 26 residues long stimulate pp34 thiophosphorylation in vitro. This stimulation pattern differs from the induction of many known defense responses in vivo, where a narrower range of smaller fragments, between approximately 10 and 15 residues long, are active. On the basis of these differences we suggest that observed effects of applied exogenous oligogalacturonides on defense responses may not necessarily reflect the situation during pathogenesis. The cell wall could act as a barrier to many exogenous oligo- and polygalacturonides as well as other large regulatory ligands.

Functional expression of low density lipoprotein receptor-related protein is controlled by receptor-associated protein in vivo.

The 39-kDa receptor-associated protein (RAP) associates with the multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) and thereby prevents the binding of all known ligands, including alpha 2-macroglobulin and chylomicron remnants. RAP is predominantly localized in the endoplasmic reticulum, raising the possibility that it functions as a chaperone or escort protein in the biosynthesis or intracellular transport of LRP. Here we have used gene targeting to show that RAP promotes the expression of functional LRP in vivo. The amount of mature, processed LRP is reduced in liver and brain of RAP-deficient mice. As a result, hepatic clearance of alpha 2-macroglobulin is impaired and remnant lipoproteins accumulate in the plasma of RAP-deficient mice that also lack functional LDL receptors. These results are consistent with the hypothesis that RAP stabilizes LRP within the secretory pathway. They also suggest a further mechanism by which the activity of an endocytic receptor may be modulated in vivo.

Auxins induce clustering of the auxin-binding protein at the surface of maize coleoptile protoplasts.

The predominant localization of the major auxin-binding protein (ABP1) of maize is within the lumen of the endoplasmic reticulum. Nevertheless, all the electrophysiological evidence supporting a receptor role for ABP1 implies that a functionally important fraction of the protein must reside at the outer face of the plasma membrane. Using methods of protoplast preparation designed to minimize proteolysis, we report the detection of ABP at the surface of maize coleoptile protoplasts by the technique of silver-enhanced immunogold viewed by epipolarization microscopy. We also show that ABP clusters following auxin treatment and that this response is temperature-dependent and auxin-specific.

Rapid endocytosis of a G protein-coupled receptor: substance P evoked internalization of its receptor in the rat striatum in vivo.

Studies on cultured cells have shown that agonists induce several types of G protein-coupled receptors to undergo internalization. We have investigated this phenomenon in rat striatum, using substance P (SP)-induced internalization of the SP receptor (SPR) as our model system. Within 1 min of a unilateral striatal injection of SP in the anesthetized rat, nearly 60% of the SPR-immunoreactive neurons within the injection zone display massive internalization of the SPR--i.e., 20-200 SPR+ endosomes per cell body. Within the dendrites the SPR undergoes a striking translocation from the plasma membrane to endosomes, and these dendrites also undergo a morphological reorganization, changing from a structure of rather uniform diameter to one characterized by large, swollen varicosities connected by thin fibers. In both cell bodies and dendrites the number of SPR+ endosomes returns to baseline within 60 min of SP injection. The number of neurons displaying substantial endosomal SPR internalization is dependent on the concentration of injected SP, and the SP-induced SPR internalization is inhibited by the nonpeptide neurokinin 1 receptor antagonist RP-67,580. These data demonstrate that in the central nervous system in vivo, SP induces a rapid and widespread SPR internalization in the cell bodies and dendrites and a structural reorganization of the dendrites. These results suggest that many of the observations that have been made on the internalization and recycling of G protein-coupled receptors in in vitro transfected cell systems are applicable to similar events that occur in the mammalian central nervous system in vivo.