N. Borgese - Academia.edu (original) (raw)

Papers by N. Borgese

Background: Tail-anchored (TA) proteins are a subclass of membrane proteins that carry out severa... more Background: Tail-anchored (TA) proteins are a subclass of membrane proteins that carry out several functions and that are targeted to their destination by unique post-translational pathways. Some TA proteins can insert in vitro into pure phospholipid bilayers spontaneously, i.e, without assistance from any chaperone, while others depend on the Get3/TRC40 pathway. One example of a spontaneously inserting TA protein is cytochrome b5, of which two forms are known: one targeting the Endoplasmic Reticulum (b5-ER) and the other one targeting the Mitochondria Outer Membrane (b5-RR). Observations: Microinjection of the recombinant b5 proteins into cells results in precise targeting of each of the two proteins, indicating that the targeting information is present in the protein. Using digitonin semi-permeabilized cells and in the presence of rabbit reticulocyte lysate (RRL), we obtain faithful targeting for both forms of cyt b5 that approaches the in cellula situation. In contrast, in the absence of cytosol both forms target the mitochondria. Attempting to find the energy-dependent chaperone responsible for the ER targeting, we observed that TCR40, Hsc-70 and Snd2 pathways play only a modest role. Recently, we found that Eyearestatin I, a p97 inhibitor, strongly inhibits the delivery of b5-ER to the ER, as monitored by glycosylation of an engineered N-glycosylation site near the C-terminus, while having no effect on a TRC40-dependent substrate. Hence, the effect seems to be specific for spontaneously inserted tail-anchored proteins, but the exact mechanism is still under investigation. Conclusions: Our results demonstrate that cyt b5 is precisely targeted to the ER by pathways distinct from the one mediated by Get3/TRC40. Since interference with TRC40 and Snd2 only minimally affect b5 targeting, it appears that multiple redundant mechanisms underlie the final specific targeting of this spontaneously inserting TA protein

Journal of Cell Science, 2002

Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal... more Tail-anchored (TA) proteins, which are defined by an N-terminal cytosolic region and a C-terminal transmembrane domain (TMD), provide useful models for studying the role of the TMD in sorting within the exo-endocytic system. Previous work has shown that a short TMD is required to keep ER-resident TA proteins from escaping to downstream compartments of the secretory pathway. To investigate the role of the TMD in TA protein sorting, we used model constructs, which consisted of GFP linked at its C-terminus to the tail region of cytochrome b(5) with TMDs of differing length or hydrophobicity. Expression of these constructs in CV-1 cells demonstrated that the feature determining exit from the ER is hydrophobicity and that if exit occurs, at least a part of the protein reaches the cell surface. To investigate which pathway to the surface is followed by plasma-membrane-directed TA constructs, we expressed the TA constructs in polarised Madin Darby Canine Kidney (MDCK) cells. The constructs...

D. 4a-Molecular mechanisms of mutant SOD1 toxicity in ALS cellular models D. 4b-Role of the VAPB ... more D. 4a-Molecular mechanisms of mutant SOD1 toxicity in ALS cellular models D. 4b-Role of the VAPB Gene in the pathogenesis of ALS

VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-... more VAPB (vesicle-associated membrane protein-associated protein B) is an endoplasmic reticulum (ER)-resident tail-anchored adaptor protein involved in lipid transport. A dominantly inherited mutant, P56S-VAPB, causes a familial form of amyotrophic lateral sclerosis (ALS) and forms poorly characterized inclusion bodies in cultured cells. To provide a cell biological basis for the understanding of mutant VAPB pathogenicity, we investigated its biogenesis and the inclusions that it generates. Translocation assays in cell-free systems and in cultured mammalian cells were used to investigate P56S-VAPB membrane insertion, and the inclusions were characterized by confocal imaging and electron microscopy. We found that mutant VAPB inserts post-translationally into ER membranes in a manner indistinguishable from the wild-type protein but that it rapidly clusters to form inclusions that remain continuous with the rest of the ER. Inclusions were induced by the mutant also when it was expressed at levels comparable to the endogenous wild-type protein. Ultrastructural analysis revealed that the inclusions represent a novel form of organized smooth ER (OSER) consisting in a limited number of parallel cisternae (usually 2 or 3) interleaved by a ϳ30 nm-thick electron-dense cytosolic layer. Our results demonstrate that the ALS-linked VAPB mutant causes dramatic ER restructuring that may underlie its pathogenicity in motoneurons.

European Journal of Biochemistry, 1980

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron ... more Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation. Electron microscopy of infected and non-infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible. Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane-bound ribosomes was produced by infection. Approximately 30% of the ribosomes and 50% of the viral RNA were found to be associated with membranes. Of the membrane-associated viral RNA, 70 % was recovered as 26-S RNA. Similar results were obtained with fibroblasts infected by the temperature-sensitive Sindbis mutant ts2, which is defective in the co-translational processing of the viral gene products at the nonpermissive temperature. Sucrose gradient analysis of membrane-bound polyribosomes solubilized by detergent indicated that as much as 50 % of the membrane-associated viral 26-S RNA is not integrated into polyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits. Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA-membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane-bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients. These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome-independent, salt-sensitive link.

FEBS Letters, 1993

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine... more Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b 5 reductase and cytochrome b 5 were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b 5 reductase and cytochrome b 5 , expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent K m values similar to those found in human liver microsomes and specific activities (V max) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b 5 in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b 5 reductase and cytochrome b 5 significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b 5 reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b 5 reductase and cytochrome b 5 in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Current Opinion in Cell Biology, 2007

A large group of diverse, functionally important, and differently localized transmembrane protein... more A large group of diverse, functionally important, and differently localized transmembrane proteins shares a particular membrane topology, consisting of a cytosolic N-terminal region, followed by a transmembrane domain close to the C-terminus. Because of their structure, these C-tail-anchored (TA) proteins must insert into all their target membranes by post-translational pathways. Recent work, based on the development of stringent and sensitive biochemical assays, has demonstrated that novel unexplored mechanisms underlie these post-translational targeting and membrane insertion pathways. Unravelling these pathways will shed light on the biosynthesis and regulation of an important group of membrane proteins and is likely to lead to new concepts in the field of membrane biogenesis.

BJOG: An International Journal of Obstetrics and Gynaecology, 1983

Journal of Biological Chemistry, Oct 5, 1992

Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three in... more Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three independent probands of hereditary methemoglobinemia type I. Patients in Kagoshima and Okinawa in Japan were shown to possess the same base change, from guanine to adenine at codon 57, which results in amino acid substitution from Arg to Gln. This nucleotide change was the same as formerly found in a patient in Toyoake, Japan (Katsube, T., Sakamoto, N., Kobayashi, Y., Seki, R., Hirano, M., Tanishima, K., Tomoda, A., Takazakura, E., Yubisui, T., Takeshita, M., Sakaki, Y., and Fukumaki, Y. (1991) Am. J. Hum. Genet. 48, 799-808). A type I patient in Italy was shown to have a base change from guanine to adenine at codon 105 which causes substitution from Val to Met. To characterize the enzymes of type I patients, Arg-57----Gln and Val-105----Met mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity. kcat/Km values (NADH) of these two enzymes were 25% in Arg-57----Gl...

$Research paper thumbnail of Heterogeneity of Golgi fractions from rat liver$

Italian Journal of Biochemistry, 1978

Cell Biology International Reports, 1990

Methods in cell biology, 1981

Publisher Summary Although the idea of a partial overlapping in the composition of the various ty... more Publisher Summary Although the idea of a partial overlapping in the composition of the various types of cellular membranes is now widely accepted, only a few multimembrane components have been adequately characterized. This chapter summarizes data regarding the in vivo and in vitro experiments on the biogenesis, membrane insertion, and turnover of one such component, the flavoprotein nicotinamide adenine dinucleotide (NADH)–cytochrome b 5 reductase located in microsomes in liver, in the Golgi complex, and in outer mitochondria1 membranes. Special emphasis is placed on the methodological aspects of these works. The relevance of findings for understanding membrane biogenesis in secretory systems is discussed. In the process of membrane biogenesis, different membrane proteins use at least two different mechanisms to become membrane-bound and to reach their final destination: cotranslational insertion, typical of some ectoproteins, and direct insertion that can occur concomitantly in several membranes without the need for transport from rough endoplasmic reticulum (ER) to other compartments. Two separate processes are believed to account for the replacement of cellular proteins and structures: autophagocytosis—by which portions of the cytoplasm are first segregated and then digested in bulk by lysosomal hydrolases—and molecular turnover.

Symposia of the Society for Experimental Biology, 1979

The EMBO journal, 1983

RNA extracted from a free polysome fraction from rat liver was used to direct translation in nucl... more RNA extracted from a free polysome fraction from rat liver was used to direct translation in nuclease-treated rabbit reticulocyte lysates, and the [35S]methionine-labelled, in vitro-synthesized, cytochrome b5 reductase was isolated with specific antibodies. Analysis by SDS-polyacrylamide gel electrophoresis, non-equilibrium pH gradient electrophoresis and one-dimensional peptide mapping failed to reveal any difference between the in vitro-synthesized reductase and the enzyme endogenous to rat liver microsomes. To study the integration of the in vitro-synthesized reductase into membranes, carboxypeptidase Y was used as a proteolytic probe. The reductase endogenous to rat liver microsomes was resistant to attack by carboxypeptidase Y, but was degraded to a smaller form when the microsomes were solubilized by detergent. Likewise, the enzyme synthesized in vitro was attacked by carboxypeptidase Y, but became largely resistant after post-translational incubation with dog pancreatic micro...

Blood, 1998

Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active i... more Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active in methemoglobin reduction, and a ubiquitous membrane-associated form involved in lipid metabolism, are produced from one gene. In the rat, the two forms are generated from alternative transcripts differing in the first exon, however, biogenesis of human b5R was less understood. Recently, two different transcripts (M and S), differing in the first exon were also described in humans. Here, we have investigated the tissue-specificity and the role of the S-transcript in the generation of soluble b5R. By RNase protection assays designed to simultaneously detect alternative b5R transcripts in the same sample, the S transcript was undetectable in nonerythroid and in erythroleukemic K562 cells induced to differentiate, but was present in terminal erythroblast cultures, and represented a major b5R transcript in reticulocytes. Analysis of the translation products of the M- and S-transcripts in HeL...

The Journal of biological chemistry, Jan 5, 1993

Two forms of cytochrome b5 are present in rat tissues, with a sequence identity of approximately ... more Two forms of cytochrome b5 are present in rat tissues, with a sequence identity of approximately 60% in the cytoplasmically exposed, tryptic fragments (Lederer, F., Ghrir, R., Guiard, B., Cortial, S., and Ito, A. (1983) Eur. J. Biochem. 132, 95-102). It has been suggested that the two isoforms have partially overlapping subcellular distributions, with each form localized to some extent on both endoplasmic reticulum and outer mitochondrial membranes (Ito, A. (1980) J. Biochem. (Tokyo) 87, 73-80). To investigate the degree of specificity of the localization of cytochrome b5 isoforms, we studied their subcellular distributions with antipeptide antibodies, one specific for microsomal cytochrome b5, one specific for outer membrane cytochrome b, and one against a sequence common to the two cytochromes. We first identified outer membrane Cyt b as a tightly bound, Triton X-114-extractable, 23-kDa polypeptide. We then analyzed biochemically characterized rat liver subcellular fractions by We...

American journal of human genetics, 1995

Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b5 reductase (b5R) (t... more Hereditary methemoglobinemia with generalized deficiency of NADH-cytochrome b5 reductase (b5R) (type II) is a rare disease characterized by severe developmental abnormalities, which often lead to premature death. Although the molecular relationship between the symptoms of this condition and the enzyme deficit are not understood, it is thought that an important cause is the loss of the lipid metabolizing activities of the endoplasmic reticulum-located reductase. However, the functions of the form located on outer mitochondrial membranes have not been considered previously. In this study, we have analyzed the gene of an Italian patient and identified a novel G-->T transversion at the splice-acceptor site of the 9th exon, which results in the complete absence of immunologically detectable b5R in blood cells and skin fibroblasts. In cultured fibroblasts of the patient, NADH-dependent cytochrome c reductase, ferricyanide reductase, and semidehydroascorbate reductase activities were se...

The Journal of biological chemistry, Jan 25, 1982

The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by ra... more The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by radioimmunoassay, using a rabbit antibody against the cathepsin D cleaved water-soluble fragment of rat liver microsomal reductase (I-reductase), which is known to be immunologically similar to the red cell enzyme. Erythrocytes contained approximately 30 ng of reductase/mg of protein, of which 90% were recovered in the hemolysate supernatant and 2.3% in the ghost fraction. After concentration by precipitation with 70% saturated (NH4)2SO4, the NADH-cytochrome c reductase activity of the soluble enzyme could be assayed in the presence of cytochrome b5, and was found to be inhibited by anti 1-reductase antibodies. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobilities of erythrocyte membrane-associated and soluble reductase of the liver microsomal enzyme and its cathepsin D cleaved hydrophilic fragment (I-reductase) were examined in crude fractions by blotting followed by sp...

The Biochemical journal, Jan 15, 1986

The intracellular localization of the post-translationally inserted integral membrane protein, NA... more The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial oute...