Isolation of a Chinese hamster ovary cell mutant defective in intramitochondrial transport of phosphatidylserine

A CHO-K1 cell mutant with a specific decrease in cellular phosphatidylethanolamine (PE) level was isolated as a variant resistant to Ro09–0198, a PE-directed antibiotic peptide. The mutant was defective in the phosphatidylserine (PS) decarboxylation pathway for PE formation, in which PS produced in the endoplasmic reticulum is transported to mitochondria and then decarboxylated by an inner mitochondrial membrane enzyme, PS decarboxylase. Neither PS formation nor PS decarboxylase activity was reduced in the mutant, implying that the mutant is defective in some step of PS transport. The transport processes of phospholipids between the outer and inner mitochondrial membrane were analyzed by use of isolated mitochondria and two fluorescence-labeled phospholipid analogs, 1-palmitoyl-2-{N-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-PS (C6-NBD-PS) and C6-NBD-phosphatidylcholine (C6-NBD-PC). On incubation with the CHO-K1 mitochondria, C6-NBD-PS was readily decarboxylated to C6-NBD-PE, suggesting that the PS analog was partitioned into the outer leaflet of mitochondria and then translocated to the inner mitochondrial membrane. The rate of decarboxylation of C6-NBD-PS in the mutant mitochondria was reduced to ≈40% of that in the CHO-K1 mitochondria. The quantity of phospholipid analogs translocated from the outer leaflet of mitochondria into inner mitochondrial membranes was further examined by selective extraction of the analogs from the outer leaflet of mitochondria. In the mutant mitochondria, the translocation of C6-NBD-PS was significantly reduced, whereas the translocation of C6-NBD-PC was not affected. These results indicate that the mutant is defective in PS transport between the outer and inner mitochondrial membrane and provide genetic evidence for the existence of a specific mechanism for intramitochondrial transport of PS.

Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

Differential Regulation of Secretory Compartments Containing the Insulin-responsive Glucose Transporter 4 in 3T3-L1 Adipocytes

Insulin and guanosine-5′-O-(3-thiotriphosphate) (GTPγS) both stimulate glucose transport and translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes. Previous studies suggest that these effects may be mediated by different mechanisms. In this study we have tested the hypothesis that these agonists recruit GLUT4 by distinct trafficking mechanisms, possibly involving mobilization of distinct intracellular compartments. We show that ablation of the endosomal system using transferrin-HRP causes a modest inhibition (∼30%) of insulin-stimulated GLUT4 translocation. In contrast, the GTPγS response was significantly attenuated (∼85%) under the same conditions. Introduction of a GST fusion protein encompassing the cytosolic tail of the v-SNARE cellubrevin inhibited GTPγS-stimulated GLUT4 translocation by ∼40% but had no effect on the insulin response. Conversely, a fusion protein encompassing the cytosolic tail of vesicle-associated membrane protein-2 had no significant effect on GTPγS-stimulated GLUT4 translocation but inhibited the insulin response by ∼40%. GTPγS- and insulin-stimulated GLUT1 translocation were both partially inhibited by GST-cellubrevin (∼50%) but not by GST-vesicle-associated membrane protein-2. Incubation of streptolysin O-permeabilized 3T3-L1 adipocytes with GTPγS caused a marked accumulation of Rab4 and Rab5 at the cell surface, whereas other Rab proteins (Rab7 and Rab11) were unaffected. These data are consistent with the localization of GLUT4 to two distinct intracellular compartments from which it can move to the cell surface independently using distinct sets of trafficking molecules.

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8,911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.

Involvement of the Ubiquitin/Proteasome System in Sorting of the Interleukin 2 Receptor β Chain to Late Endocytic Compartments

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor β chain (IL2Rβ) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rβ was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rβ, with little effect on its internalization. In addition, we show that a fraction of IL2Rβ can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rβ targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rβ were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rβ toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Subcellular imaging of intramitochondrial Ca2+ with recombinant targeted aequorin: significance for the regulation of pyruvate dehydrogenase activity.

Specific targeting of the recombinant, Ca2+ -sensitive photoprotein, aequorin to intracellular organelles has provided new insights into the mechanisms of intracellular Ca2+ homeostasis. When applied to small mammalian cells, a major limitation of this technique has been the need to average the signal over a large number of cells. This prevents the identification of inter- or intracellular heterogeneities. Here we describe the imaging in single mammalian cells (CHO.T) of [Ca2+] with recombinant chimeric aequorin targeted to mitochondria. This was achieved by optimizing expression of the protein through intranuclear injection of cDNA and through the use of a charge-coupled device camera fitted with a dual microchannel plate intensifier. This approach allows accurate quantitation of the kinetics and extent of the large changes in mitochondrial matrix [Ca2+] ([Ca2+](m)) that follow receptor stimulation and reveal different behaviors of mitochondrial populations within individual cells. The technique is compared with measurements of [Ca2+](m) using the fluorescent indicator, rhod2. Comparison of [Ca2+](m) with the activity of the Ca2+ -sensitive matrix enzyme, pyruvate dehydrogenase (PDH), reveals that this enzyme is a target of the matrix [Ca2+] changes. Peak [Ca2+](m) values following receptor stimulation are in excess of those necessary for full activation of PDH in situ, but may be necessary for the activation of other mitochondrial dehydrogenases. Finally, the data suggest that the complex regulation of PDH activity by a phosphorylation-dephosphorylation cycle may provide a means by which changes in the frequency of cytosolic (and hence mitochondrial) [Ca2+] oscillations can be decoded by mitochondria.

Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

Overexpression of thioredoxin in transgenic mice attenuates focal ischemic brain damage

Thioredoxin (TRX) plays important biological roles both in intra- and extracellular compartments, including in regulation of various intracellular molecules via thiol redox control. We produced TRX overexpressing mice and confirmed that there were no anatomical and physiological differences between wild-type (WT) mice and TRX transgenic (Tg) mice. In the present study we subjected mice to focal brain ischemia to shed light on the role of TRX in brain ischemic injury. At 24 hr after middle cerebral artery occlusion, infarct areas and volume were significantly smaller in Tg mice than in WT mice. Moreover neurological deficit was ameliorated in Tg mice compared with WT mice. Protein carbonyl content, a marker of cellular protein oxidation, in Tg mice showed less increase than did that of WT mice after the ischemic insult. Furthermore, c-fos expression in Tg mice was stronger than in WT mice 1 hr after ischemia. Our results suggest that transgene expression of TRX decreased ischemic neuronal injury and that TRX and the redox state modified by TRX play a crucial role in brain damage during stroke.

Malignant transformation of early lymphoid progenitors in mice expressing an activated Blk tyrosine kinase

The intracellular signals governing cellular proliferation and developmental progression during lymphocyte development are incompletely understood. The tyrosine kinase Blk is expressed preferentially in the B lineage, but its function in B cell development has been largely unexplored. We have generated transgenic mice expressing constitutively active Blk [Blk(Y495F)] in the B and T lymphoid compartments. Expression of Blk(Y495F) in the B lineage at levels similar to that of endogenous Blk induced B lymphoid tumors of limited clonality, whose phenotypes are characteristic of B cell progenitors at the proB/preB-I to preB-II transition. Expression of constitutively active Blk in the T lineage resulted in the appearance of clonal, thymic lymphomas composed of intermediate single positive cells. Taken together, these results indicate that specific B and T cell progenitor subsets are preferentially susceptible to transformation by Blk(Y495F) and suggest a role for Blk in the control of proliferation during B cell development.

A chloroplast processing enzyme functions as the general stromal processing peptidase

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.

Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation

We present an approach for monitoring protein–protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing β-galactosidase (β-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the β-gal activity resulting from the forced interaction of nonfunctional weakly complementing β-gal peptides (Δα and Δω) serves as a measure of the extent of interaction of the non-β-gal portions of the chimeras. To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Δα and Δω, respectively. Enzymatic β-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.

MLN64 contains a domain with homology to the steroidogenic acute regulatory protein (StAR) that stimulates steroidogenesis

MLN64 is a protein that is highly expressed in certain breast carcinomas. The C terminus of MLN64 shares significant homology with the steroidogenic acute regulatory protein (StAR), which plays a key role in steroid hormone biosynthesis by enhancing the intramitochondrial translocation of cholesterol to the cholesterol side-chain cleavage enzyme. We tested the ability of MLN64 to stimulate steroidogenesis by using COS-1 cells cotransfected with plasmids expressing the human cholesterol side-chain cleavage enzyme system and wild-type and mutant MLN64 proteins. Wild-type MLN64 increased pregnenolone secretion in this system 2-fold. The steroidogenic activity of MLN64 was found to reside in the C terminus of the protein, because constructs from which the C-terminal StAR homology domain was deleted had no steroidogenic activity. In contrast, removal of N-terminal sequences increased MLN64’s steroidogenesis-enhancing activity. MLN64 mRNA was found in many human tissues, including the placenta and brain, which synthesize steroid hormones but do not express StAR. Western blot analysis revealed the presence of lower molecular weight immunoreactive MLN64 species that contain the C-terminal sequences in human tissues. Homologs of both MLN64 and StAR were identified in Caenorhabditis elegans, indicating that the two proteins are ancient. Mutations that inactivate StAR were correlated with amino acid residues that are identical or similar among StAR and MLN64, indicating that conserved motifs are important for steroidogenic activity. We conclude that MLN64 stimulates steroidogenesis by virtue of its homology to StAR.

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis thaliana

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [γ-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.