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M Pilon

Publications and source records attributed to M Pilon.

At least 19 recordsLinked to original sources
PubMed2000

The protein translocation channel mediates glycopeptide export across the endoplasmic reticulum membrane.

Peptides and misfolded secretory proteins are transported efficiently from the endoplasmic reticulum (ER) lumen to the cytosol, where the proteins are degraded by proteasomes. Protein export depends on Sec61p, the ribosome-binding core component of the protein translocation channel in the ER membrane. We found that prebinding of ribosomes abolished export of a glycopeptide from yeast microsomes. Deletion of SSH1, which encodes a ribosome-binding Sec61p homologue in the ER, had no effect on glycopeptide export. A collection of cold-sensitive sec61 mutants displayed a variety of phenotypes: two mutants strongly defective in misfolded protein export from the ER, sec61-32 and sec61-41, displayed only minor peptide export defects. Glycopeptide export was severely impaired, however, in several sec61 mutants that were only marginally defective in misfolded protein export. In addition, a mutation in SEC63 strongly reduced peptide export from the ER. ER-luminal ATP was required for both misfolded protein and glycopeptide export. We conclude that the protein translocation channel in the ER membrane mediates glycopeptide transport across the ER membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Breeding mercury-breathing plants for environmental cleanup.

In an elegant study, Richard Meagher's research group has succeeded in introducing a bacterial mercury detoxification pathway into plants. The resulting plants show enhanced mercury tolerance and can convert highly toxic forms of organic mercury to less toxic elemental mercury, which is volatile. The significance of this study is that it could lead to the more efficient and affordable cleanup of environmental mercury pollution, and in a broader context, it proves the power of genetic engineering for phytoremediation.

Environmental Pollutants↗
PubMed2000

The diabetes autoantigen ICA69 and its Caenorhabditis elegans homologue, ric-19, are conserved regulators of neuroendocrine secretion.

ICA69 is a diabetes autoantigen with no homologue of known function. Given that most diabetes autoantigens are associated with neuroendocrine secretory vesicles, we sought to determine if this is also the case for ICA69 and whether this protein participates in the process of neuroendocrine secretion. Western blot analysis of ICA69 tissue distribution in the mouse revealed a correlation between expression levels and secretory activity, with the highest expression levels in brain, pancreas, and stomach mucosa. Subcellular fractionation of mouse brain revealed that although most of the ICA69 pool is cytosolic and soluble, a subpopulation is membrane-bound and coenriched with synaptic vesicles. We used immunostaining in the HIT insulin-secreting beta-cell line to show that ICA69 localizes in a punctate manner distinct from the insulin granules, suggesting an association with the synaptic-like microvesicles found in these cells. To pursue functional studies on ICA69, we chose to use the model organism Caenorhabditis elegans, for which a homologue of ICA69 exists. We show that the promoter of the C. elegans ICA69 homologue is specifically expressed in all neurons and specialized secretory cells. A deletion mutant was isolated and found to exhibit resistance to the drug aldicarb (an inhibitor of acetylcholinesterase), suggesting defective neurotransmitter secretion in the mutant. On the basis of the aldicarb resistance phenotype, we named the gene ric-19 (resistance to inhibitors of cholinesterase-19). The resistance to aldicarb was rescued by introducing a ric-19 transgene into the ric-19 mutant background. This is the first study aimed at dissecting ICA69 function, and our results are consistent with the interpretation that ICA69/RIC-19 is an evolutionarily conserved cytosolic protein participating in the process of neuroendocrine secretion via association with certain secretory vesicles.

Amino Acid Sequence↗
PubMed1998

Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane.

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles of sec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61 mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression of SEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

Adenosine Triphosphate↗
PubMed1998

Domains of a transit sequence required for in vivo import in Arabidopsis chloroplasts.

Nuclear-encoded precursors of chloroplast proteins are synthesized with an amino-terminal cleavable transit sequence, which contains the information for chloroplastic targeting. To determine which regions of the transit sequence are most important for its function, the chloroplast uptake and processing of a full-length ferredoxin precursor and four mutants with deletions in adjacent regions of the transit sequence were analyzed. Arabidopsis was used as an experimental system for both in vitro and in vivo import. The full-length wild-type precursor translocated efficiently into isolated Arabidopsis chloroplasts, and upon expression in transgenic Arabidopsis plants only mature-sized protein was detected, which was localized inside the chloroplast. None of the deletion mutants was imported in vitro. By analyzing transgenic plants, more subtle effects on import were observed. The most N-terminal deletion resulted in a fully defective transit sequence. Two deletions in the middle region of the transit sequence allowed translocation into the chloroplast, although with reduced efficiencies. One deletion in this region strongly reduced mature protein accumulation in older plants. The most C-terminal deletion was translocated but resulted in defective processing. These results allow the dissection of the transit sequence into separate functional regions and give an in vivo basis for a domain-like structure of the ferredoxin transit sequence.

Amino Acid Sequence↗
PubMed1997

Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.

Degradation of misfolded secretory proteins has long been assumed to occur in the lumen of the endoplasmic reticulum (ER). Recent evidence, however, suggests that such proteins are instead degraded by proteasomes in the cytosol, although it remains unclear how the proteins are transported out of the ER. Here we provide the first genetic evidence that Sec61p, the pore-forming subunit of the protein translocation channel in the ER membrane, is directly involved in the export of misfolded secretory proteins. We describe two novel mutants in yeast Sec61p that are cold-sensitive for import into the ER in both intact yeast cells and a cell-free system. Microsomes derived from these mutants are defective in exporting misfolded secretory proteins. These proteins become trapped in the ER and are associated with Sec61p. We conclude that misfolded secretory proteins are exported for degradation from the ER to the cytosol via channels formed by Sec61p.

Alleles↗
PubMed1997

Early events leading to fate decisions during leech embryogenesis.

This paper reviews leech development up to the 12-cell embryo. Oogenesis proceeds by a system of nurse cells that contribute to oocyte growth via continuous cytoplasmic connections. Development begins when fertilized eggs are deposited: formation of the polar bodies, and centration of the male and female pro-nuclei is accompanied by cytoskeletal contractions, and formation of teloplasm (yolk-free cytoplasm). The first cleavages are asymmetric: cell D', the largest macromere in the eight-cell embryo, contains most of the teloplasm. At fourth cleavage D' divides equally; its animal and vegetal daughters are precursors of segmental ectoderm and mesoderm, respectively. Teloplasm is a determinant of the D' cell fate. The expression pattern of Hro-nos, a leech homolog to the Drosophila gene nanos, suggests that it may be a determinant associated with the animal cortex and inducing the ectodermal fate in the animal daughter cell of the D' macromere.

Journal Article↗
PubMed1997

A nanos homolog in leech.

From the glossiphoniid leech Helobdella robusta, we have cloned and determined the complete coding sequence of Hro-nos, a gene homologous to the nanos gene from Drosophila melanogaster. Developmental northern blots show that Hro-nos, like nanos, is a maternal transcript that decays rapidly during early development. A polyclonal antiserum raised against the HRO-NOS protein was used in developmental western blots and for immunostaining leech embryos of different developmental stages. The HRO-NOS protein is first detectable in 2-cell embryos (4-6 hours of development) and exhibits a transient expression peaking during fourth cleavage (9-12 cells; 8-14 hours of development). The HRO-NOS protein exhibits a graded distribution along the primary embryonic axis and is partitioned unequally between the sister cells DNOPQ and DM, progeny of macromere D' at fourth cleavage: DNOPQ is the segmental ectoderm precursor cell and exhibits levels of HRO-NOS protein that are at least two-fold higher than in cell DM, the segmental mesoderm precursor cell. The observed expression pattern suggests that Hro-nos plays a role in the decision between ectodermal and mesodermal cell fates in leech.

Amino Acid Sequence↗
PubMed1996

Motor profile of patients in minimally responsive and persistent vegetative states.

The purpose of this descriptive clinical study is to document the motor characteristics of patients in minimally responsive and persistent vegetative states. Twelve subjects, presenting a prolonged altered state of consciousness (x = 7.82 years, range 2-27), aged 27-78 years (x = 50, SD = 15.26) were evaluated, using standardized protocols, on the following variables: passive range of motion, observed movements, reflexes, tonus, postural status and reactions. The subjects' level of awareness and responsivity were measured with the Coma/Near Coma (CNC) scale at each of the three data collection sessions. While group CNC scores were stable over the three sessions, fluctuations in the level of awareness of individual subjects was recorded, confirming the heterogeneity of this clientele. Abnormal primitive reflexes were present in all subjects, with the flexor withdrawal (75%), the tonic labyrinthine (36%) and the body-on-body righting reaction (25%) being the most frequently observed. All subjects presented altered tonus, considerable posturing and varied degrees of reduced range of joint motion. A range of abnormal (e.g. chewing, clonus) and normal patterned (e.g. bridging, scratching) movement behaviours was recorded, but these did not translate into functional use, such as rolling. Collectively, the findings stress the complexity of the motor profile of patients in minimally responsive and vegetative states, and suggest the need for physiotherapists to become more actively involved in the evaluation and treatment of this clientele.

Adult↗
PubMed1995

Functional domains of the ferredoxin transit sequence involved in chloroplast import.

In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wild-type precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.

Amino Acid Sequence↗
PubMed1995

Chloroplast import and sequential maturation of pea carbonic anhydrase: the roles of various parts of the transit peptide.

Chloroplast pea carbonic anhydrase is synthesised in the cytosol with an unusually long bipartite N-terminal extension of the mature sequence previously proposed to serve as a transit peptide. Studies of import into pea chloroplasts show that the N-terminal 69 amino acids of the previously proposed transit peptide is sufficient for translocation and localisation to the stroma, while the acidic C-terminal part does not seem to have any function in these processes. Processing of the in vitro imported precursors is shown to be at a new cleavage site located in the middle of the actual transit peptide. The results indicate that maturation occurs in more than one step. The time-course does not seem to be dependent on the age of the chloroplast but on the age of the translocated precursor.

Carbonic Anhydrases↗
PubMed1994

Precursor-mediated opening of translocation pores in chloroplast envelopes.

Direct electrical measurements on native chloroplast envelopes reveal that a full-length chloroplast precursor protein causes an increase in the conductivity of the envelope membranes, due to its transit sequence. The conductivity is not influenced by a truncated precursor protein incapable of efficient translocation, suggesting precursor-mediated opening of translocation pores in chloroplast envelopes.

Chloroplasts↗
PubMed1993

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids.

Import of proteins into chloroplasts depends on an N-terminal transit sequence. Transit sequences contain little primary sequence similarity and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of the transit sequence, we studied the transit peptide of preferredoxin (trfd) by circular dichroism. In buffer, trfd is in a random coil conformation. A large increase in alpha-helix was induced in the presence of micelles or vesicles formed by anionic lipids. Less pronounced changes in secondary structure were induced by zwitterionic detergents but no changes were observed in the presence of neutral detergents or vesicles composed of phosphatidylcholine.

Chloroplasts↗
PubMed1993

The transit sequence mediates the specific interaction of the precursor of ferredoxin with chloroplast envelope membrane lipids.

The interaction of the precursor of the chloroplast protein ferredoxin with membrane lipids was studied in monolayer experiments in order to investigate the possible involvement of membrane lipids in the protein translocation process. The precursor efficiently and specifically inserts into a total lipid extract of its biological target the outer envelope membrane of chloroplasts. This interaction is mediated by the transit sequence as it can also be observed for the chemically prepared transit peptide of ferredoxin but neither for the ferredoxin apoprotein nor holoprotein. Interactions with the individual chloroplast lipids, monogalactosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, and phosphatidylglycerol are predominantly involved which corresponds to the results obtained for transit peptide fragments of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (van't Hof, R., Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol and phosphatidylcholine, suggesting that a loose lipid headgroup packing due to small lipid headgroups and/or electrostatic repulsions facilitates efficient insertion. The observed preferences for interaction of the precursor and transit peptide of ferredoxin for the chloroplast outer envelope membrane lipid extract and the presequence of cytochrome c oxidase subunit IV for the mitochondrial outer membrane lipid extract indicate that targeting sequence-lipid interactions contribute to organelle-specific protein targeting.

Amino Acid Sequence↗
PubMed1992

Secondary structure and folding of a functional chloroplast precursor protein.

Ferredoxin is a chloroplast stroma protein which is cytosolically synthesized as a precursor with an amino-terminal extension called the transit sequence that is needed for the post-translational uptake by the chloroplast. To characterize the secondary and tertiary structure elements, the full precursor, the holo- and apo- (without iron-sulfur cluster) forms of the mature protein, and the chemically synthesized transit peptide were obtained and analyzed separately. Circular dichroism, tryptophan fluorescence quenching, and protease accessibility experiments indicate that the precursor has a low content of defined secondary structure and resembles unfolded proteins; these properties are due to both the mature part and the transit sequence. This result provides an explanation for the lack of cytosolic factor requirement of this protein for import. In an import competition assay, the isolated transit peptide had an affinity for the chloroplasts comparable to the full precursor. Interestingly and of possible importance to the import process, the transit peptide has conformational flexibility as it adopts alternative secondary structures in different environments.

Amino Acid Sequence↗