Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Fatty acid synthesis in chloroplasts is regulated by light. The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark. Plants have ACCase in plastids and the cytosol. To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant. Only the plastidic ACCase was activated by DTT. This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone. Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m. The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase. These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle. The catalytic activity of ACCase was maximum at pH 8 and 2–5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity. These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration. A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

A Cell Signal Pathway Involving Laminin-5, α3β1 Integrin, and Mitogen-activated Protein Kinase Can Regulate Epithelial Cell Proliferation

Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.

The Win1 Mitotic Regulator Is a Component of the Fission Yeast Stress-activated Sty1 MAPK Pathway

The fission yeast Sty1 mitogen-activated protein (MAP) kinase (MAPK) and its activator the Wis1 MAP kinase kinase (MAPKK) are required for cell cycle control, initiation of sexual differentiation, and protection against cellular stress. Like the mammalian JNK/SAPK and p38/CSBP1 MAPKs, Sty1 is activated by a range of environmental insults including osmotic stress, hydrogen peroxide, UV light, menadione, heat shock, and the protein synthesis inhibitor anisomycin. We have recently identified two upstream regulators of the Wis1 MAPKK, namely the Wak1 MAPKKK and the Mcs4 response regulator. Cells lacking Mcs4 or Wak1, however, are able to proliferate under stressful conditions and undergo sexual differentiation, suggesting that additional pathway(s) control the Wis1 MAPKK. We now show that this additional signal information is provided, at least in part, by the Win1 mitotic regulator. We show that Wak1 and Win1 coordinately control activation of Sty1 in response to multiple environmental stresses, but that Wak1 and Win1 perform distinct roles in the control of Sty1 under poor nutritional conditions. Our results suggest that the stress-activated Sty1 MAPK integrates information from multiple signaling pathways.

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.

Active Nucleocytoplasmic Shuttling Required for Function and Regulation of Stress-Activated Kinase Spc1/StyI in Fission Yeast

Transcriptional induction of many stress-response genes is dependent on stress-induced nuclear accumulation of stress-activated protein kinases (SAPKs). In the fission yeast Schizosaccharomyces pombe, nuclear accumulation of the SAPK Spc1 (also known as StyI) requires activating phosphorylation catalyzed by the SAPK kinase Wis1; however, it is unknown whether the localization of Spc1 is regulated by nuclear transport factors. Herein are reported studies that show that Spc1 localization is regulated by active transport mechanisms during osmotic stress. Nuclear import of Spc1 requires Pim1, a homologue of the guanine nucleotide exchange factor RCC1 that is essential for nucleocytoplasmic shuttling of proteins. Nuclear export of Spc1 is regulated by the export factor Crm1. An Spc1–Crm1 complex forms as Spc1 is exported from the nucleus. Wis1 and the tyrosine phosphatases Pyp1 and Pyp2 that inactivate Spc1 are excluded from the nucleus by a Crm1-independent mechanism; hence the nuclear import of Spc1 leads to transient isolation from its regulatory proteins. Thus, active nucleocytoplasmic shuttling is required for both the function and regulation of Spc1 during the osmotic shock response.

Chs6p-dependent Anterograde Transport of Chs3p from the Chitosome to the Plasma Membrane in Saccharomyces cerevisiae

Chitin synthase III (CSIII), an enzyme required to form a chitin ring in the nascent division septum of Saccharomyces cerevisiae, may be transported to the cell surface in a regulated manner. Chs3p, the catalytic subunit of CSIII, requires the product of CHS6 to be transported to or activated at the cell surface. We find that chs6Δ strains have morphological abnormalities similar to those of chs3 mutants. Subcellular fractionation and indirect immunofluorescence indicate that Chs3p distribution is altered in chs6 mutant cells. Order-of-function experiments using end4–1 (endocytosis-defective) and chs6 mutants indicate that Chs6p is required for anterograde transport of Chs3p from an internal endosome-like membrane compartment, the chitosome, to the plasma membrane. As a result, chs6 strains accumulate Chs3p in chitosomes. Chs1p, a distinct chitin synthase that acts during or after cell separation, is transported normally in chs6 mutants, suggesting that Chs1p and Chs3p are independently packaged during protein transport through the late secretory pathway.

BiP and Immunoglobulin Light Chain Cooperate to Control the Folding of Heavy Chain and Ensure the Fidelity of Immunoglobulin Assembly

The immunoglobulin (Ig) molecule is composed of two identical heavy chains and two identical light chains (H2L2). Transport of this heteromeric complex is dependent on the correct assembly of the component parts, which is controlled, in part, by the association of incompletely assembled Ig heavy chains with the endoplasmic reticulum (ER) chaperone, BiP. Although other heavy chain-constant domains interact transiently with BiP, in the absence of light chain synthesis, BiP binds stably to the first constant domain (CH1) of the heavy chain, causing it to be retained in the ER. Using a simplified two-domain Ig heavy chain (VH-CH1), we have determined why BiP remains bound to free heavy chains and how light chains facilitate their transport. We found that in the absence of light chain expression, the CH1 domain neither folds nor forms its intradomain disulfide bond and therefore remains a substrate for BiP. In vivo, light chains are required to facilitate both the folding of the CH1 domain and the release of BiP. In contrast, the addition of ATP to isolated BiP–heavy chain complexes in vitro causes the release of BiP and allows the CH1 domain to fold in the absence of light chains. Therefore, light chains are not intrinsically essential for CH1 domain folding, but play a critical role in removing BiP from the CH1 domain, thereby allowing it to fold and Ig assembly to proceed. These data suggest that the assembly of multimeric protein complexes in the ER is not strictly dependent on the proper folding of individual subunits; rather, assembly can drive the complete folding of protein subunits.

Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

Uptake and Intracellular Transport of Acidic Fibroblast Growth Factor: Evidence for Free and Cytoskeleton-anchored Fibroblast Growth Factor Receptors

Endocytic uptake and intracellular transport of acidic FGF was studied in cells transfected with FGF receptor 4 (FGFR4). Acidification of the cytosol to block endocytic uptake from coated pits did not inhibit endocytosis of the growth factor in COS cells transfected with FGFR4, indicating that it is to a large extent taken up by an alternative endocytic pathway. Fractionation of the cells demonstrated that part of the growth factor receptor was present in a low-density, caveolin-containing fraction, but we were unable to demonstrate binding to caveolin in immunoprecipitation studies. Upon treatment of the cells with acidic FGF, the activated receptor, together with the growth factor, moved to a juxtanuclear compartment, which was identified as the recycling endosome compartment. When the cells were lysed with Triton X-100, 3-([3-chloramidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate, or 2-octyl glucoside, almost all surface-exposed and endocytosed FGFR4 was solubilized, but only a minor fraction of the total FGFR4 in the cells was found in the soluble fraction. The data indicate that the major part of FGFR4 is anchored to detergent-insoluble structures, presumably cytoskeletal elements associated with the recycling endosome compartment.

Integrin regulation of c-Abl tyrosine kinase activity and cytoplasmic–nuclear transport

The product of the c-abl protooncogene is a nonreceptor tyrosine kinase found in both the cytoplasm and the nucleus. We report herein that cell adhesion regulates the kinase activity and subcellular localization of c-Abl. When fibroblastic cells are detached from the extracellular matrix, kinase activity of both cytoplasmic and nuclear c-Abl decreases, but there is no detectable alteration in the subcellular distribution. Upon adhesion to the extracellular matrix protein fibronectin, a transient recruitment of a subset of c-Abl to early focal contacts is observed coincident with the export of c-Abl from the nucleus to the cytoplasm. The cytoplasmic pool of c-Abl is reactivated within 5 min of adhesion, but the nuclear c-Abl is reactivated after 30 min, correlating closely with its return to the nucleus and suggesting that the active nuclear c-Abl originates in the cytoplasm. In quiescent cells where nuclear c-Abl activity is low, the cytoplasmic c-Abl is similarly regulated by adhesion but the nuclear c-Abl is not activated upon cell attachment. These results show that c-Abl activation requires cell adhesion and that this tyrosine kinase can transmit integrin signals to the nucleus where it may function to integrate adhesion and cell cycle signals.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

Stretch-activated single K+ channels account for whole-cell currents elicited by swelling

Functionally significant stretch-activated ion channels have been clearly identified in excitable cells. Although single-channel studies suggest their expression in other cell types, their activity in the whole-cell configuration has not been shown. This discrepancy makes their physiological significance doubtful and suggests that their mechanical activation is artifactual. Possible roles for these molecules in nonexcitable cells are acute cell-volume regulation and, in epithelial cells, the complex adjustment of ion fluxes across individual cell membranes when the rate of transepithelial transport changes. We report the results of experiments on isolated epithelial cells expressing in the basolateral membrane stretch-activated K+ channels demonstrable by the cell-attached patch-clamp technique. In these cells, reversible whole-cell currents were elicited by both isosmotic and hyposmotic cell swelling. Cation selectivity and block by inorganic agents were the same for single-channel and whole-cell currents, indicating that the same entity underlies single-channel and whole-cell currents and that the single-channel events are not artifactual. In these cells, when the rate of apical-membrane NaCl entry increases, the cell Na+ content and volume also increase, stimulating the Na+,K+-ATPase at the basolateral membrane, i.e., both Na+ extrusion and K+ uptake increase. We speculate that, under these conditions, the parallel activation of basolateral K+ channels (by the swelling) elevates conductive K+ loss, tending to maintain the cell K+ content constant (“pump-leak parallelism”). This study describes a physiologically relevant stretch-activated channel, at both the single-channel and whole-cell levels, in a nonneural cell type.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

The voltage- and Ca2+-activated K+ (KV,Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel KV,Ca α-subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., KV,Ca β-subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.

Prostacyclin-mediated activation of peroxisome proliferator-activated receptor δ in colorectal cancer

There is evidence from both genetic and pharmacologic studies to suggest that the cyclooxygenase-2 (COX-2) enzyme plays a causal role in the development of colorectal cancer. However, little is known about the identity or role of the eicosanoid receptor pathways activated by COX-derived prostaglandins (PG). We previously have reported that COX-2-derived prostacyclin promotes embryo implantation in the mouse uterus via activation of the nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) δ. In light of the recent finding that PPARδ is a target of β-catenin transactivation, it is important to determine whether this signaling pathway is operative during the development of colorectal cancer. Analysis of PPARδ mRNA in matched normal and tumor samples revealed that expression of PPARδ, similar to COX-2, is up-regulated in colorectal carcinomas. In situ hybridization studies demonstrate that PPARδ is expressed in normal colon and localized to the epithelial cells at the very tips of the mucosal glands. In contrast, expression of PPARδ mRNA in colorectal tumors was more widespread with increased levels in transformed epithelial cells. Analysis of PPARδ and COX-2 mRNA in serial sections suggested they were colocalized to the same region within a tumor. Finally, transient transfection assays established that endogenously synthesized prostacyclin (PGI2) could serve as a ligand for PPARδ. In addition, the stable PGI2 analog, carbaprostacyclin, and a synthetic PPARδ agonist induced transactivation of endogenous PPARδ in human colon carcinoma cells. We conclude from these observations that PPARδ, similar to COX-2, is aberrantly expressed in colorectal tumors and that endogenous PPARδ is transcriptionally responsive to PGI2. However, the functional consequence of PPARδ activation in colon carcinogenesis still needs to be determined.