Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways

Vesicles formed from endoplasmic reticulum (ER) by a cell-free system of leek cells (Allium porrum) are enriched in phosphatidylserine (PS), especially species containing very long chain fatty acids (VLCFA, at least 20 carbon atoms). In plant cells, PS is formed either by PS synthase or the serine exchange enzyme, although it is not known which pathway(s) contribute(s) to PS delivery in the ER-derived vesicles (EV), nor to what extent this occurs. Taking advantage of a cell-free system, we have shown that PS enrichment originates mainly from the serine exchange enzyme which is the only pathway that synthesizes the VLCFA-PS species. On the other hand, both enzymes synthesize PS with long chain fatty acids (up to 18 carbon atoms), but these species are given to the EV by PS synthase.     

Phosphoenolpyruvate Carboxykinase Is Involved in the Decarboxylation of Aspartate in the Bundle Sheath of Maize

We recently showed that maize (Zea mays L.) leaves contain appreciable amounts of phosphoenolpyruvate carboxykinase (PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood [1997] Aust J Plant Physiol 24: 459–468). In the present study, we investigated the role of PEPCK in C₄ photosynthesis in maize. PEPCK activity and protein were enriched in extracts from bundle-sheath (BS) strands compared with whole-leaf extracts. Decarboxylation of [4-¹⁴C]aspartate (Asp) by BS strands was dependent on the presence of 2-oxoglutarate and Mn²⁺, was stimulated by ATP, was inhibited by the PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent of illumination. The principal product of Asp metabolism was phosphoenolpyruvate, whereas pyruvate was a minor product. Decarboxylation of [4-¹⁴C]malate was stimulated severalfold by Asp and 3-phosphoglycerate, was only slightly reduced in the absence of Mn²⁺ or in the presence of 3-mercaptopicolinic acid, and was light dependent. Our data show that decarboxylation of Asp and malate in BS cells of maize occurs via two different pathways: Whereas malate is mainly decarboxylated by NADP-malic enzyme, decarboxylation of Asp is dependent on the activity of PEPCK.     

The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.     

Chemical synthesis and translesion replication of a cis-syn cyclobutane thymine-uracil dimer

The cytosine base in DNA undergoes hydrolytic deamination at a considerable rate when UV radiation induces formation of a cyclobutane pyrimidine dimer (CPD) with an adjacent pyrimidine base. We have synthesized a phosphoramidite building block of a cis–syn cyclobutane thymine–uracil dimer (T[]U), which is the deaminated form of the CPD at a TC site, and incorporated it into oligodeoxyribonucleotides. The previously reported method for synthesis of the thymine dimer (T[]T) was applied, using partially protected thymidylyl-(3′–5′)-2′-deoxyuridine as the starting material, and after triplet- sensitized irradiation, the configuration of the base moiety in the major product was determined by NMR spectroscopy. Presence of the cis–syn cyclobutane dimer in the obtained oligonucleotides was confirmed by UV photoreversal and reaction with T4 endonuclease V. Using a 30mer containing T[]U, translesion synthesis by human DNA polymerase η was analyzed. There was no difference in the results between the templates containing T[]T and T[]U and pol η bypassed both lesions with the same efficiency, incorporating two adenylates. This enzyme showed fidelity to base pair formation, but this replication causes a C→T transition because the original sequence is TC.     

Survival of Enterococcus faecalis in an Oligotrophic Microcosm: Changes in Morphology, Development of General Stress Resistance, and Analysis of Protein Synthesis

The ability of Enterococcus faecalis to metabolically adapt to an oligotrophic environment has been analyzed. E. faecalis is able to survive for prolonged periods under conditions of complete starvation established by incubation in tap water. During incubation in this microcosm, cells developed a rippled cell surface with irregular shapes. Exponentially growing cells survived to the same extent as cells starved for glucose prior to exposure to the multiple nutrient deficient stress. Chloramphenicol treatment during incubation in tap water led to a rapid decline in plate counts for exponentially growing cells but showed progressively reduced influence on stationary-phase cells harvested after different times of glucose starvation. During incubation in the oligotrophic environment, cells from the exponential-growth phase and early-stationary phase became progressively more resistant to other environmental stresses (heat [62°C], acid [pH 3.3], UV_{254 nm} light [180 J/m²], and sodium hypochlorite [0.05%]) until they reached a maximum of survival characteristic for each treatment. In contrast, cells starved of glucose for 24 h did not become more resistant to the different treatments during incubation in tap water. Our combined data suggest that energy starvation induces a response similar to that triggered by oligotrophy. Analysis of protein synthesis by two-dimensional gel electrophoresis revealed the enhanced synthesis of 51 proteins which were induced in the oligotrophic environment. A comparison of these oligotrophy-inducible proteins with the 42 glucose starvation-induced polypeptides (J. C. Giard, A. Hartke, S. Flahaut, P. Boutibonnes, and Y. Auffray, Res. Microbiol. 148:27–35, 1997) showed that 16 are common between the two different starvation conditions. These proteins and the corresponding genes seem to play a key role in the observed phenomena of long-term survival and development of general stress resistance of starved cultures of E. faecalis.     

Coupled ER to Golgi Transport Reconstituted with Purified Cytosolic Proteins

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-α-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi transport even in the presence of saturating levels of purified fusion factors. Manipulation of the semi-intact cell assay is used to distinguish freely diffusible ER- derived vesicles containing pro-α-factor from docked vesicles and from fused vesicles. Uso1p mediates vesicle docking and produces a dilution resistant intermediate. Sec18p and LMA1 are not required for the docking phase, but are required for efficient fusion of ER- derived vesicles with the Golgi complex. Surprisingly, elevated levels of Sec23p complex (a subunit of the COPII coat) prevent vesicle fusion in a reversible manner, but do not interfere with vesicle docking. Ordering experiments using the dilution resistant intermediate and reversible Sec23p complex inhibition indicate Sec18p action is required before LMA1 function.     

ADP-Glucose Pyrophosphorylase from Potato Tubers. Site-Directed Mutagenesis Studies of the Regulatory Sites

Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706–24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107–24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg²⁺. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.     

Morphological and Functional Association of Sec22b/ERS-24 with the pre-Golgi Intermediate Compartment

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER–Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER–Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15°C is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER–Golgi transport.     

In vitro and in vivo modulations of benzo[c]phenanthrene-DNA adducts by DNA mismatch repair system

Benzo[c]phenanthrene dihydrodiol epoxide (B[c] PhDE) is well known as an important environmental chemical carcinogen that preferentially modifies DNA in adenine residues. However, the molecular mechanism by which B[c]PhDE induces tumorigenesis is not fully understood. In this report, we demonstrate that DNA mismatch repair (MMR), a genome maintenance system, plays an important role in B[c]PhDE-induced carcinogensis by promoting apoptosis in cells treated with B[c]PhDE. We show that purified human MMR recognition proteins, MutSα and MutSβ, specifically recognized B[c]PhDE-DNA adducts. Cell lines proficient in MMR exhibited several-fold more sensitivity to killing than cell lines defective in either MutSα or MutLα by B[c]PhDE; the nature of this sensitivity was shown to be due to increased apoptosis. Additionally, wild-type mice exposed to B[c]PhDE had intestinal crypt cells that underwent apoptosis significantly more often than intestinal crypt cells found in B[c]PhDE-treated Msh2^–/– or Mlh1^–/– mice. These findings, combined with previous studies, suggest that the MMR system may serve as a general sensor for chemical-caused DNA damage to prevent damaged cells from mutagenesis and carcinogenesis by promoting apoptosis.     

Chloroplast-avoidance response induced by high-fluence blue light in prothallial cells of the fern adiantum capillus-veneris as analyzed by microbeam irradiation

Chloroplast movement was induced by partial cell illumination using a high-fluence blue microbeam in light-grown and dark-adapted prothallial cells of the fern Adiantum capillus-veneris. Chloroplasts inside the illuminated area moved out (high-fluence response [HFR]), whereas those outside moved toward the irradiated area (low-fluence response [LFR]), although they stopped moving when they reached the border. These results indicate that both HFR and LFR signals are generated by high-fluence blue light of the same area, and that an LFR signal can be transferred long-distance from the beam spot, although an HFR signal cannot. The lifetime of the HFR signal was calculated from the traces of chloroplast movement induced by a brief pulse from a high-fluence blue microbeam to be about 6 min. This is very short compared with that of the LFR (30–40 min; T. Kagawa, M. Wada [1994] J Plant Res 107: 389–398). These data indicate that the signal transduction pathways of the HFR and the LFR must be distinct.     

Thank ORP9 for FFAT: With endosomal ORP10, it’s fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Most cellular lipids are synthesized in the ER, often undergoing rapid redistribution to other cellular membranes, thereby maintaining low concentrations at the ER. Consequently, lipids exiting the ER may need to be transported against their concentration gradient. Lipid flow along a gradient to the ER can drive countertransport of ER-derived lipid to membranes with a higher lipid concentration. This nonvesicular lipid exchange occurs at membrane contact sites (MCS), where different organelles are closely apposed, providing a platform for lipid transport proteins including oxysterol-binding protein (OSBP)-related proteins (ORPs). Lipid specificity, which varies between ORPs, is defined by the OSBP-related domain (ORD). The ORD of ORP10 shares phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) binding residues with ORP5/8 and can bind and extract PS from liposomes (1), suggesting a potential role in PI4P-PS counter transport, analogous to that of ORP5/8 at ER–plasma membrane MCS (2). ORPs are targeted to specific organelles by interaction between their PH domain and membrane phospholipids. Most ORPs also possess a FFAT motif (two phenylalanines in an acidic tract), which simultaneously targets the ORP to ER-localized VAMP-associated proteins (VAPs) at MCS between the ER and other organelles. ORP10, however, lacks a FFAT motif, yet was found to stabilize ER–Golgi MCSs (Fig. 1 A) for PI4P transport to the ER (2). Kawasaki et al. have now uncovered a novel function for ORP10 in PI4P–PS lipid exchange at the ER–endosome interface (Fig. 1 B), with downstream effects on endosomal fission and retrograde transport (3).Open in a separate windowFigure 1.Regulation of retrograde and secretory traffic by ORP10-mediated lipid exchange. (A) ORP10 interacts with VAP-bound ORP9 at ER–endosome and ER–Golgi MCSs, with downstream effects on retrograde transport of mannose 6-phosphate receptor (M6PR). Boxed region (detailed in B) depicts ORP10 at the ER–endosome interface. (B) ORP10 functions in lipid exchange between the ER and endosomes, transporting endosomal PI4P to the ER in exchange for ER-derived PS. Production of PI4P in endosomes by PI4KIIα-dependent phosphorylation of phosphatidylinositol (PI), coupled with its consumption in the ER by ER-localized Sac1, generates a PI4P concentration gradient from the endosome to the ER. Low membrane PS concentrations in the ER are maintained by PS inhibition of PS synthesis from phosphatidylcholine (PC) by Pss1 or from PE by Pss2, with PS synthesis at ER–endosome contact sites promoting rapid PS export from the ER in yeast (not yet known if a similar mechanism operates in mammalian cells). ORP10 mediates PI4P transport along its gradient to the ER, driving countertransport of PS by ORP10 against its concentration gradient to the endosome. PS enrichment at the endosome leads to recruitment of the ATPase EHD1 to facilitate endosome fission for retrograde transport. (C) Depletion of ORP10 prevents lipid exchange at ER–endosome contact sites, resulting in a loss of retrograde transport of M6PR. Additionally, ER–Golgi MCSs are diminished, and secretion of ApoB-100 is increased.The PH domain of ORP10 selectively binds PI4P and is required for ORP10 recruitment to the TGN (2) and endosomes (3), both home to PI4KIIα, a PI4P-producing kinase. Rapid PI4P degradation at the ER by the phosphatase Sac1 generates a PI4P gradient at the ER–endosome or TGN interface, with PI4P flow to the ER driving countertransport of PS to the endosome (as also predicted for the Golgi). Activity of endosomal PI4P phosphatase Sac2 (4) may hamper formation of an endosome–ER PI4P gradient, but since ORP10 did not colocalise well with Sac2 (3), they likely function at different endosome populations.PS synthesis at MCSs may also contribute to ORP10-mediated lipid exchange. Targeting PS synthase to ER:mitochondria contacts in yeast was found topromote PS transport out of the ER to mitochondria (5). Similarly, ER to endosome PS transport was increased when PS synthase was targeted to ER:endosome MCS. Localized PS gradients from PS synthesis in the ER at MCSs, coupled with rapid decarboxylation of PS to phosphatidylethanolamine (PE) in mitochondria/endosomes by yeast PS decarboxylases Psd1/Psd2, could contribute to lipid exchange. In mammalian cells, though, since no endosomal decarboxylase has been identified, ORP10-mediated lipid exchange is likely to be primarily driven by the PI4P gradient. Whether this process is facilitated by localized activation of PS synthase at MCS has not yet been demonstrated. Since PS synthase activity is negatively regulated by PS, exit from the ER is a key factor in its biogenesis. Recruitment of specific ORPs to endosomes/TGN by PI4P for ER tethering and consequent lipid exchange provides an elegant regulatory pathway for PI4P–PS homeostasis in cellular membranes.ORP10 shares functional similarities with ORP11: both proteins comprise an N-terminal PH domain and a C-terminal ORD, with a linker region in between harboring a coiled-coiled domain. Unlike other ORPs, ORP10 and ORP11 possess neither a FFAT motif nor a membrane spanning domain to enable ER interaction, but heterotypic interaction with ORP9, which does contain a FFAT motif, has been demonstrated for both proteins. Kawasaki et al. identified an ORP9-ORP10 interaction at ER–endosome MCSs that is dependent on the ORP10 linker region. ORP9 was also implicated in ORP10-mediated lipid exchange at the TGN, where it may play a redundant role with OSBP in maintaining ER contact. Similarly, ORP11 is also recruited to the TGN and, to a lesser extent, the endosome, by ORP9, with the interaction depending on the linker region of both proteins (6).The finely tuned regulation of PI4P/PS is emerging as an important determinant of endocytic traffic. Previous studies have shown that endosomal PI4P accumulation inhibits retrograde transport from endosomes to the TGN (7), while endosomal PS regulates endosome to Golgi retrograde traffic. As depicted in Fig. 1 B, Kawasaki et al. have built on this to show that through interaction with VAP-bound ORP9, ORP10 mediates lipid countertransport at ER–endosome MCSs, removing PI4P from, and supplying PS to, the endosome, with consequent recruitment of the membrane scission protein EHD1 to control endosomal fission and retrograde transport (3). Spatial and temporal regulation of endosome fission by ER–endosome MCSs involves recruitment of the ER membrane protein TMCC1 to the budding endosome by the actin regulator Coronin 1C, stabilizing the MCS (7), but the mechanism by which MCS might effect scission has remained elusive. The findings of Kawaski et al. present an explanation: by providing a platform for lipid exchange, MCS promote the recruitment of EHD1, which belongs to a conserved class of ATPases that can oligomerise in ring-like structures around tubules to mediate fission (8). VAP interaction with OSBP at ER–endosome MCSs is also required for retrograde transport (7), but potential redundancy between ORP9/OSBP in ORP10-mediated lipid exchange, or if ORP10 functions at Coronin 1C/TMCC1-regulated MCS is not yet established.Interestingly, ORP10 function at the TGN has been implicated in regulating ApoB-100 secretion (Fig. 1 C), with hypersecretion reported in ORP10-depleted cells (9). FFAP1, which promotes PI4P consumption by Sac1 at ER:TGN contacts, also negatively regulates ApoB-100 exit from the TGN in a PI4KIIIβ-dependent manner, suggesting direct regulation of ApoB-100 secretion by PI4P at the TGN (9). Could PI4P coordinate nutrient sensing with cargo sorting and secretion at the TGN? PI4P has been described as lipid biosensor of cytosolic pH, with protonation of its head group regulating protein interactions (10). The influence of cytosolic pH on ORP10-PI4P interaction may provide an additional layer of regulation of lipoprotein secretion in response to changes in cellular energy/pH.How ORP10 function is coordinated at Golgi and endosomal membranes and the significance of potential redundancy with ORP11 remains unclear. The regulation of Sac2 activity and how it relates to ER-endosome lipid exchange is also intriguing. While questions still remain, an important role for ORP10 is emerging in maintaining homoeostasis between endosome maturation, retrograde traffic and secretory transport.     

Ypt1/Rab1 regulates Hrr25/CK1δ kinase activity in ER–Golgi traffic and macroautophagy

ER-derived COPII-coated vesicles are conventionally targeted to the Golgi. However, during cell stress these vesicles also become a membrane source for autophagosomes, distinct organelles that target cellular components for degradation. How the itinerary of COPII vesicles is coordinated on these pathways remains unknown. Phosphorylation of the COPII coat by casein kinase 1 (CK1), Hrr25, contributes to the directional delivery of ER-derived vesicles to the Golgi. CK1 family members are thought to be constitutively active kinases that are regulated through their subcellular localization. Instead, we show here that the Rab GTPase Ypt1/Rab1 binds and activates Hrr25/CK1δ to spatially regulate its kinase activity. Consistent with a role for COPII vesicles and Hrr25 in membrane traffic and autophagosome biogenesis, hrr25 mutants were defective in ER–Golgi traffic and macroautophagy. These studies are likely to serve as a paradigm for how CK1 kinases act in membrane traffic.     

Lysophosphatidic Acid Acyltransferase from Coconut Endosperm Mediates the Insertion of Laurate at the sn-2 Position of Triacylglycerols in Lauric Rapeseed Oil and Can Increase Total Laurate Levels

Expression of a California bay laurel (Umbellularia californica) 12:0-acyl-carrier protein thioesterase, bay thioesterase (BTE), in developing seeds of oilseed rape (Brassica napus) led to the production of oils containing up to 50% laurate. In these BTE oils, laurate is found almost exclusively at the sn-1 and sn-3 positions of the triacylglycerols (T.A. Voelker, T.R. Hayes, A.C. Cranmer, H.M. Davies [1996] Plant J 9: 229–241). Coexpression of a coconut (Cocos nucifera) 12:0-coenzyme A-preferring lysophosphatitic acid acyltransferase (D.S. Knutzon, K.D. Lardizabal, J.S. Nelsen, J.L. Bleibaum, H.M. Davies, J.G. Metz [1995] Plant Physiol 109: 999–1006) in BTE oilseed rape seeds facilitates efficient laurate deposition at the sn-2 position, resulting in the acccumulation of trilaurin. The introduction of the coconut protein into BTE oilseed rape lines with laurate above 50 mol % further increases total laurate levels.     

Biogenesis of gamma-secretase early in the secretory pathway

γ-Secretase is responsible for proteolytic maturation of signaling and cell surface proteins, including amyloid precursor protein (APP). Abnormal processing of APP by γ-secretase produces a fragment, Aβ₄₂, that may be responsible for Alzheimer's disease (AD). The biogenesis and trafficking of this important enzyme in relation to aberrant Aβ processing is not well defined. Using a cell-free reaction to monitor the exit of cargo proteins from the endoplasmic reticulum (ER), we have isolated a transient intermediate of γ-secretase. Here, we provide direct evidence that the γ-secretase complex is formed in an inactive complex at or before the assembly of an ER transport vesicle dependent on the COPII sorting subunit, Sec24A. Maturation of the holoenzyme is achieved in a subsequent compartment. Two familial AD (FAD)–linked PS1 variants are inefficiently packaged into transport vesicles generated from the ER. Our results suggest that aberrant trafficking of PS1 may contribute to disease pathology.     

Transport of Axl2p Depends on Erv14p,an ER–Vesicle Protein Related to the Drosophila cornichon Gene Product

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER–vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not efficiently delivered to the cell surface. Axl2p is required for selection of axial growth sites and normally localizes to nascent bud tips or the mother bud neck. In erv14Δ strains, Axl2p accumulates in the ER while other secretory proteins are transported at wild-type rates. We propose that Erv14p is required for the export of specific secretory cargo from the ER. The polarity defect of erv14Δ yeast cells is reminiscent of cornichon mutants, in which egg chambers fail to establish proper asymmetry during early stages of oogenesis. These results suggest an unforeseen conservation in mechanisms producing cell polarity shared between yeast and Drosophila.     

Inhibition of poly(ADP-ribose) polymerase activity is insufficient to induce tetraploidy

Poly(ADP-ribose) polymerase (PARP) knockout mice are resistant to murine models of human diseases such as cerebral and myocardial ischemia, traumatic brain injury, diabetes, Parkinsonism, endotoxic shock and arthritis, implicating PARP in the pathogenesis of these diseases. Potent selective PARP inhibitors are therefore being evaluated as novel therapeutic agents in the treatment of these diseases. Inhibition or depletion of PARP, however, increases genomic instability in cells exposed to genotoxic agents. We recently demonstrated the presence of a genomically unstable tetraploid population in PARP^–/– fibroblasts and its loss after stable transfection with PARP cDNA. To elucidate whether the genomic instability is attributable to PARP deficiency or lack of PARP activity, we investigated the effects of PARP inhibition on development of tetraploidy. Immortalized wild-type and PARP^–/– fibroblasts were exposed for 3 weeks to 20 µM GPI 6150 (1,11b-dihydro-[2H]benzopyrano[4,3,2-de]isoquinolin-3-one), a novel small molecule specific competitive inhibitor of PARP (K_i = 60 nM) and one of the most potent PARP inhibitors to date (IC₅₀ = 0.15 µM). Although GPI 6150 initially decreased cell growth in wild-type cells, there was no effect on cell growth or viability after 24 h. GPI 6150 inhibited endogenous PARP activity in wild-type cells by ~91%, to about the residual levels in PARP^–/– cells. Flow cytometric analysis of unsynchronized wild-type cells exposed for 3 weeks to GPI 6150 did not induce the development of tetraploidy, suggesting that, aside from its catalytic function, PARP may play other essential roles in the maintenance of genomic stability.     

Cytomegalovirus Assembly Protein Precursor and Proteinase Precursor Contain Two Nuclear Localization Signals That Mediate Their Own Nuclear Translocation and That of the Major Capsid Protein

The cytomegalovirus (CMV) assembly protein precursor (pAP) interacts with the major capsid protein (MCP), and this interaction is required for nuclear translocation of the MCP, which otherwise remains in the cytoplasm of transfected cells (L. J. Wood et al., J. Virol. 71:179–190, 1997). We have interpreted this finding to indicate that the CMV MCP lacks its own nuclear localization signal (NLS) and utilizes the pAP as an NLS-bearing escort into the nucleus. The CMV pAP amino acid sequence has two clusters of basic residues (e.g., KRRRER [NLS1] and KARKRLK [NLS2], for simian CMV) that resemble the simian virus 40 large-T-antigen NLS (D. Kalderon et al., Cell 39:499–509, 1984) and one of these (NLS1) has a counterpart in the pAP homologs of other herpesviruses. The work described here establishes that NLS1 and NLS2 are mutually independent NLS that can act (i) in cis to translocate pAP and the related proteinase precursor (pNP1) into the nucleus and (ii) in trans to transport MCP into the nucleus. By using combinations of NLS mutants and carboxy-terminal deletion constructs, we demonstrated a self-interaction of pAP and cytoplasmic interactions of pAP with pNP1 and of pNP1 with itself. The relevance of these findings to early steps in capsid assembly, the mechanism of MCP nuclear transport, and the possible cytoplasmic formation of protocapsomeric substructures is discussed.     

Vacuolation: Origin and development of the lysosomal apparatus in root-tip cells

Summary The morphology of vacuolation has been investigated in root tip cells of corn using the freeze-etching technique. The genesis of vacuoles involves the following processes: a) Formation of small, endoplasmic-reticulum (ER)-derived vesicles (provacuoles); b) fusion of provacuoles resulting in the formation of small vacuoles, and followed by fusion and expansion of vacuoles; c) incorporation of large, dictyosome-derived vesicles into vacuoles by invagination of the tonoplast; d) invagination of the tonoplast resulting in the incorporation of cytoplasmic material into vacuoles. The morphological findings are correlated with biochemical data obtained from isolated vacuoles (lysosomes). Provacuoles (ER-derived vesicles) are shown to be primary lysosomes; their hydrolases arise from the ER. Vacuoles represent secondary lysosomes (digestive vacuoles) of the higher-plant cell. The metabolic role of lytic processes proceeding in the lysosomal apparatus is discussed.     

Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability

A key issue in the nucleotide excision repair (NER) of bulky carcinogen–DNA adducts is the ability of the NER machinery to recognize and repair certain adducts while failing to repair others. Unrepaired adducts can survive to cause mutations that initiate the carcinogenic process. Benzo[c]phenanthrene (B[c]Ph), a representative fjord region polycyclic aromatic hydrocarbon, can be metabolically activated to the enantiomeric benzo[c]phenanthrene diol epoxides (B[c]PhDEs), (+)-(1S,2R,3R,4S)-3,4- dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phe nanthrene and the corresponding (–)-(1R,2S,3S,4R) isomer. These react predominantly with adenine residues in DNA to produce the stereoisomeric 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts. Duplexes containing the 1R (+) or 1S (–) B[c]Ph-dA adduct in codon 61 of the human N-ras mutational hotspot sequence CA*A, with B[c]Ph modification at A*, are not repaired by the human NER system. However, the analogous stereoisomeric DNA adducts of the bay region benzo[a]pyrene diol epoxide (B[a]PDE), 10S (+)- and 10R (–)-trans-anti-B[a]P-N⁶-dA, are repaired in the same base sequence. In order to elucidate structural and thermodynamic origins of this phenomenon, we have carried out a 2 ns molecular dynamics simulation for the 1R (+)- and 1S (–)-trans-anti-B[c]Ph-N⁶-dA adducts in an 11mer duplex containing the human N-ras codon 61 sequence, and compared these results with our previous study of the B[a]P-dA adducts in the same sequence. The molecular mechanics Poisson– Boltzmann surface area (MM-PBSA) method was applied to calculate the free energies of the pair of stereoisomeric B[c]Ph-dA adducts, and a detailed structural analysis was carried out. The different repair susceptibilities of the B[a]P-dA adducts and the B[c]Ph-dA adducts can be attributed to different degrees of distortion, stemming from combined effects of differences in the quality of Watson–Crick hydrogen bonding, unwinding, stretching and helix backbone perturbations. These differences are due to the different intrinsic topologies of the rigid, planar bay region adducts versus the twisted, sterically hindered fjord region adducts.