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M Colombini

Publications and source records attributed to M Colombini.

At least 37 records · Page 2Linked to original sources
PubMed1997

Isolation and characterization of the mitochondrial channel, VDAC, from the insect Heliothis virescens.

A 31 kDa voltage-dependent anion-selective channel (VDAC) protein was purified from the insect Heliothis virescens (tobacco budworm, denoted TBW) using an alkali extraction and filtration procedure and was characterized by SDS-PAGE, amino acid sequencing, biophysical properties and immunocytochemistry. The N-terminal sequence has highest identity with VDACs from mammals (50-66%) followed by plants (34-41%) and lower eukaryotes (30-34%). Reconstitution in planar phospholipid membranes yielded properties typical of VDACs from other organisms including a single-channel conductance of 4.1 nS (in 1 M KCl), closure in response to positive and negative transmembrane voltage, and a reversal potential of 11.8 mV indicating anion selectivity in the open state. A polyclonal antiserum (R19) raised against gel-purified 31 kDa protein specifically labelled mitochondria and mitochondrial outer membranes in TBW flight muscle by light and electron microscope immunocytochemistry.

Amino Acid Sequence↗
PubMed1997

Regulation of metabolite flux through voltage-gating of VDAC channels.

The mitochondrial outer membrane channel, VDAC, is thought to serve as the major permeability pathway for metabolite flux between the cytoplasm and mitochondria. The permeability of VDAC to citrate, succinate, and phosphate was studied in channels reconstituted into planar phospholipid membranes. All ions showed large changes in permeability depending on whether the channel was in the open or in the low conductance, "closed" state, with the closed state always more cation selective. This was especially true for the divalent and trivalent anions. Additionally, the anion flux when the voltage was zero was shown to decrease to 5-11% of the open state flux depending on the anion studied. These results give the first rigorous examination of the ability of metabolites to permeate through VDAC channels and indicate that these channels can control the flux of these ions through the outer membrane. This lends more evidence to the growing body of experiments that suggest that the outer mitochondrial membrane has a much more important role in controlling mitochondrial activity than has been thought historically.

Ion Channel Gating↗
PubMed1997

VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function.

The mitochondrial channel, VDAC, forms large (3 nm in diameter) aqueous pores through membranes. We measured ATP flow (using the luciferin/luciferase method) through these channels after reconstitution into planar phospholipid membranes. In the open state of VDAC, as many as 2 x 10(6) ATP molecules can flow through one channel per second. The half-maximum rate occurs at approximately 75 mM ATP. The permeability of a single channel for ATP is 1.1 x 10(-14) cm3/s (about 1 cm/s after correcting for cross-sectional area), which is 100 times less than the permeability for chloride and 10 times less than that for succinate. Channel closure results in a 50% reduction in conductance, showing that monovalent ions are still quite permeable, yet ATP flux is almost totally blocked. This is consistent with an electrostatic barrier that results in inversion of the selectivity of the channel and could be an example of how large channels selectively control the flow of charged metabolites. Thus VDAC is ideally suited to controlling the flow of ATP between the cytosol and the mitochondrial spaces.

Adenosine Triphosphate↗
PubMed1997

Autodirected insertion: preinserted VDAC channels greatly shorten the delay to the insertion of new channels.

VDAC, a mitochondrial outer membrane channel, has the ability to catalyze and direct the insertion of other VDAC channels into planar phospholipid membranes. The spontaneous rate of insertion of detergent-solubilized VDAC channels into phospholipid membranes is estimated to be 1.5 x 10(-5) channels min-1 micron-2. VDAC channels already in the membrane can increase this rate by a factor of 10(9). The presence of 5 M urea on the opposite side of the membrane increases this 10-fold to 4.5 x 10(5) channels min-1 microns-2. Similar but weaker effects are observed with Triton X100 addition (10(-3)% (v/v)). These agents are not acting on uninserted channels because they do not affect the delay from sample addition to first insertion. Under the chosen conditions, this delay is long (240 s) without preinserted channels. However, the presence of a few VDAC channels in the membrane reduces this delay to 14 s, close to the diffusion limit. Therefore, urea and Triton, added to the side of the membrane opposite that to which the VDAC sample was added, likely increase the flexibility of the VDAC channels in the membrane, allowing them to be more efficient catalysts for VDAC insertion. There are obvious implications for membrane protein insertion and targeting.

Cell Membrane↗
PubMed1997

Multicopy suppressors of phenotypes resulting from the absence of yeast VDAC encode a VDAC-like protein.

The permeability of the outer mitochondrial membrane to most metabolites is believed to be based in an outer membrane, channel-forming protein known as VDAC (voltage-dependent anion channel). Although multiple isoforms of VDAC have been identified in multicellular organisms, the yeast Saccharomyces cerevisiae has been thought to contain a single VDAC gene, designated POR1. However, cells missing the POR1 gene (delta por1) were able to grow on yeast media containing a nonfermentable carbon source (glycerol) but not on such media at elevated temperature (37 degrees C). If VDAC normally provides the pathway for metabolites to pass through the outer membrane, some other protein(s) must be able to partially substitute for that function. To identify proteins that could functionally substitute for POR1, we have screened a yeast genomic library for genes which, when overexpressed, can correct the growth defect of delta por1 yeast grown on glycerol at 37 degrees C. This screen identified a second yeast VDAC gene, POR2, encoding a protein (YVDAC2) with 49% amino acid sequence identity to the previously identified yeast VDAC protein (YVDAC1). YVDAC2 can functionally complement defects present in delta por1 strains only when it is overexpressed. Deletion of the POR2 gene alone had no detectable phenotype, while yeasts with deletions of both the POR1 and POR2 genes were viable and able to grow on glycerol at 30 degrees C, albeit more slowly than delta por1 single mutants. Like delta por1 single mutants, they could not grow on glycerol at 37 degrees C. Subcellular fractionation studies with antibodies which distinguish YVDAC1 and YVDAC2 indicate that YVDAC2 is normally present in the outer mitochondrial membrane. However, no YVDAC2 channels were detected electrophysiologically in reconstituted systems. Therefore, mitochondrial membranes made from wild-type cells, delta por1 cells, delta por1 delta por2 cells, and delta por1 cells overexpressing YVDAC2 were incorporated into liposomes and the permeability of resulting liposomes to nonelectrolytes of different sizes was determined. The results indicate that YVDAC2 does not confer any additional permeability to these liposomes, suggesting that it may not normally form a channel. In contrast, when the VDAC gene from Drosophila melanogaster was expressed in delta por1 yeast cells, VDAC-like channels could be detected in the mitochondria by both bilayer and liposome techniques, yet the cells failed to grow on glycerol at 37 degrees C. Thus, channel-forming activity does not seem to be either necessary or sufficient to restore growth on nonfermentable carbon sources, indicating that VDAC mediates cellular functions that do not depend on the ability to form channels.

Amino Acid Sequence↗
PubMed1996

ATP flux is controlled by a voltage-gated channel from the mitochondrial outer membrane.

A voltage-gated channel, called VDAC (mitochondrial porin) is known to be responsible for most of the metabolite flux across the mitochondrial outer membrane. Here, direct measurements of ATP flux through VDAC channels reconstituted into planar phospholipid membranes establish that VDAC is sufficient to provide passage for ATP efflux from mitochondria. Further, the gating of the channel can shut down ATP flux completely while, simultaneously, allowing the flow of small ions. Thus, these channels are ideally suited to control ATP flux through the mitochondrial outer membrane and, consequently, mitochondrial function. The block to ATP flow through the closed state is likely to be not steric but electrostatic.

Adenosine Triphosphate↗
PubMed1996

The role of pyridine dinucleotides in regulating the permeability of the mitochondrial outer membrane.

Both NADH and NADPH reduce the permeability of the mitochondrial outer membrane to ADP. This is specific for the outer membrane and uncorrelated with the respiratory control ratio. This could result in a 7-fold difference between the concentration of ADP in the intermembrane space and that in the external environment (at 5 microM ADP). In both cases the permeability declines by a factor of 5, but NADH is more potent: KD = 86 microM for NADH versus 580 microM for NADPH. The lower apparent affinity for NADPH is partly explained by Mg2+-NADPH being the active species, and under our conditions only 30% of the NADPH is in this form. The corrected KD is 184 microM. Free NADH has the same charge as the Mg2+-NADPH complex, and thus both likely bind to the same site. The ability of NADH and NADPH to induce the closure of reconstituted VDAC channels is consistent with VDAC being the main pathway for metabolite flow across the outer membrane. Oncotic pressure, effective at inducing VDAC closure, also decreases the outer membrane permeability. Thus, in the presence of cytosolic colloidal osmotic pressure NAD(P)H may inhibit mitochondrial catabolic pathways and divert reducing equivalents to anabolic pathways.

Binding Sites↗
PubMed1996

Self-catalyzed insertion of proteins into phospholipid membranes.

We present evidence that the mitochondrial channel, VDAC, when reconstituted into a phospholipid membrane, can catalyze the insertion of other VDAC channels. This property called "auto-directed insertion" was first proposed by Zizi et al. (1995) to explain observations on asymmetric VDAC channels. We found that 2 urea or guanidinium chloride (GdmCl) caused a burst of insertions of VDAC channels when added to the same side as VDAC addition. More strikingly, when added to the opposite side they caused a 10-60-fold sustained yet reversible increase in insertion rate. Protein stabilization by sarcosine eliminated the effect of urea and GdmCl on VDAC insertion. Control experiments showed that water flow, ionic strength, osmotic force, phospholipid type, and membrane potential were not involved. Therefore, although both urea and GdmCl affect the properties of phospholipid membranes, it is more likely that these agents act either by changing the structure of the pre-inserted channels, allowing them to be more effective catalysts for VDAC insertion, or by flowing through the channels and acting on nearby VDAC channels inducing them to insert. Either way, insertion must be occurring next to pre-inserted channels. Urea and GdmCl may mimic chaperones by partially unfolding VDAC and keeping it in an insertion-competent state. "Auto-directed insertion" may ensure both correct targeting and orientation of nascent proteins in vivo.

Catalysis↗
PubMed1996

Individual leaflets of a membrane bilayer can independently regulate permeability.

Water rapidly crosses most membranes, but only slowly crosses apical membranes of barrier epithelia such as bladder and kidney collecting duct, a feature essential to barrier function. How apical membrane structure reduces permeabilities remains unclear. Cell plasma membranes contain two leaflets of distinct lipid composition; the role of this bilayer asymmetry in membrane permeability is unclear. To determine how asymmetry of leaflet composition affects membrane permeability, effects on bilayer permeation of reducing single leaflet permeability were determined using two approaches: formation of asymmetric bilayers in an Ussing chamber, with only one of two leaflets containing cholesterol sulfate, and stabilization of the external leaflet of unilamellar vesicles with praeseodymium (Pr3+). In both systems, permeability measurements showed that each leaflet acts as an independent resistor of water permeation. These results show that a single bilayer leaflet can act as the barrier to permeation and provide direct evidence that segregation of lipids to create a low permeability of barrier epithelial apical membranes.

Cell Membrane Permeability↗
PubMed1996

The control of mitochondrial respiration in yeast: a possible role of the outer mitochondrial membrane.

Mitochondrial respiration in yeast (S. cerevisiae) is regulated by the level of glucose in the medium. Glucose is known to inhibit respiration by repressing key enzymes in the respiratory chain. We present evidence that the early events in this inhibition include the closure of VDAC channels, the primary pathway for metabolite flow across the outer membrane. Aluminum hydroxide is known to inhibit the closure of VDAC. Addition of aluminum acetylacetonate to yeast cells, which should elevate the aluminum hydroxide concentrations in the cytoplasm, caused the inhibition of cell respiration by glucose to be delayed for up to 100 min. No significant effect of aluminum was observed in cells grown on glycerol. Yeast cells lacking the VDAC gene were also unresponsive to the addition of aluminum salt in the presence of glucose. Therefore, the closure of VDAC channels may be an early step in the inhibition of the respiration of yeast by glucose.

Aluminum Hydroxide↗
PubMed1996

Indications of a common folding pattern for VDAC channels from all sources.

Previous research on the mitochondrial channel VDAC from the yeast S. cerevisiae had identified protein strands forming the wall of VDAC's aqueous pore. Here we report the results of analyzing the primary sequences of VDAC from various sources to see if the transmembrane folding pattern identified from this yeast is conserved for VDAC of different species. We analyzed the primary sequences of VDAC from higher plants, fungi, invertebrates, and vertebrates and found that all have a very similar "beta-pattern" profile with 12-15 peaks indicating potential sided beta strands that are candidates for protein strands forming the wall of the aqueous pore. All these VDAC sequences can be put into the 13 transmembrane strand folding pattern previously identified for yeast VDAC. These folding patterns agree with available experimental data: both electrophysiological and protease digestion data. Although the primary sequences of VDAC from very diverse organisms show low homology, sequence similarity in the proposed corresponding 13 transmembrane strands is substantial. Competing proposals utilizing 16 transmembrane beta strands are in conflict with electrophysiological experimental observations and violate the constraints on such strands, such as no charged amino acids facing the phospholipid membrane and sufficient number of residues to span the membrane.

Amino Acid Sequence↗
PubMed1996

Biliopancreatic diversion for obesity at eighteen years.

BACKGROUND: Surgical attempts to treat obesity began because of the discouraging results of conservative medical treatment, which successfully achieved initial weight loss but failed to maintain it. Gastric restrictive procedures, currently the most popular surgical methods for obesity therapy, have proved to be effective in initiating weight loss, but some concerns regarding their long-term efficacy in weight maintenance have arisen. METHODS: Of a total of 1968 obese patients who underwent biliopancreatic diversion since 1976, the last consecutive 1217 underwent the "ad hoc stomach" type of diversion with a 200 cm alimentary limb, a 50 cm common limb, and a gastric volume varying between 200 and 500 ml. Mean age was 37 years old (11 to 69 years), and mean excess weight was 117%. Maximum follow-up was 115 months with nearly 100% participation. RESULTS: In the last half of the series, operative mortality was 0.4% with no general complications and with early surgical complications of wound dehiscence and infection (total, 1.2%) and late complications of incisional hernia (8.7%) and intestinal obstruction (1.2%). Mean percent loss initial excess weight (IEW) at 2, 4, 6, and 8 years was 78 +/- 16, 75 +/- 16, 78 +/- 18, and 77 +/- 16 in the patients with IEW up to 120% and 74 +/- 12, 73 +/- 13, 73 +/- 12, and 72 +/- 10 in those with IEW more than 120%. A group of 40 patients who underwent the original "half-half" biliopancreatic diversion maintained a mean 70% reduction of IEW during a 15-year follow-up period. Specific late complications included anemia (less than 5%), stomal ulcer (2.8%), protein malnutrition (7% with 1.7% requiring surgical revision by common limb elongation or by restoration). Clinical problems from bone demineralization were minimal in the short term and almost absent in the long term. CONCLUSIONS: Biliopancreatic diversion is a very effective procedure but is potentially dangerous if used incorrectly.

Adolescent↗
PubMed1995

Oriented channel insertion reveals the motion of a transmembrane beta strand during voltage gating of VDAC.

Yeast VDAC channels (isolated from the mitochondrial outer membrane) form large aqueous pores whose walls are believed to consist of 1 alpha helix and 12 beta strands. Each channel has two voltage-gating processes: one closes the channels at positive potentials, the other at negative. When VDAC is reconstituted into phospholipid (soybean) membranes, the two gating processes have virtually the same steepness of voltage dependence and the same midpoint voltage. Substituting lysine for glutamate at either end of one putative beta strand (E145K or E152K) made the channels behave asymmetrically, increasing the voltage dependence of one gating process but not the other. The asymmetry was the same whether 1 or 100 channels were in the membrane, indicating oriented channel insertion. However, the direction of insertion varied from membrane to membrane, indicating that the insertion of the first channel was random and subsequent insertions were directed by the previously inserted channel(s). This raises the prospect of an auto-directed insertion with possible implications to protein targeting in cells. Each of the mutations affected a different gating process because the double mutant increased voltage dependence of both processes. Thus this strand may slide through the membrane in one direction or the other depending on the gating process. We propose that the model of folding for VDAC be altered to move this strand into the sensor region of the protein where it may act as a tether and guide/restrict the motion of the sensor.

Amino Acid Sequence↗
PubMed1994

Beta-NADH decreases the permeability of the mitochondrial outer membrane to ADP by a factor of 6.

Mitochondria with intact outer membrane (99% intact based on cytochrome c impermeability) were isolated and used to measure the permeability of their outer membrane to ADP. beta-NADH reduced the permeability in a concentration-dependent manner (KD = 87 +/- 5 microM) by a factor of 6. alpha-NADH and beta-NAD+ cannot mimic the action of beta-NADH. The mitochondrial outer membranes become rate-limiting in the presence of beta-NADH at low, physiologically relevant, ADP concentrations (< 30 microM). beta-NADH has been shown to increase the voltage dependence of VDAC (a major pathway for metabolite transport across the outer membrane) in a reconstituted system and this may be the way it acts on the isolated mitochondria. Inhibition of beta-NADH dehydrogenases does not inhibit the action of beta-NADH indicating that it is not acting by delivering reducing equivalents. The ability of beta-NADH, produced by glycolysis, to inhibit mitochondrial function by reducing the permeability of the outer membrane may be one pathway responsible for the Crabtree effect.

Adenosine Diphosphate↗
PubMed1994

Characterization and partial purification of the VDAC-channel-modulating protein from calf liver mitochondria.

The mitochondrial channel, VDAC, mediates metabolic flux across the mitochondrial outer membrane. When reconstituted into planar phospholipid membranes, VDAC is voltage-dependent, existing in multiple conformational states with different selectivities and permeabilities. At low membrane potentials, these channels are in the open state and are anion-selective. VDAC channels switch to lower-conductive closed states at high membrane potentials. The VDAC modulator, a soluble mitochondrial protein, has been demonstrated to dramatically increase the voltage dependence of VDAC channels and induce the channels to enter closed states even at low membrane potentials. We have isolated and partially purified this modulating protein and the activity is associated with a 54 kDa protein on SDS-PAGE. Under native reduced conditions the activity eluted around 100 kDa from a gel filtration column. As little as 200 ng/ml of the partially purified protein was sufficient to modulate reconstituted VDAC channels. This protein had a pI of 5.1. A second activity with a pI of 4.8 was far more potent, making VDAC-channel-containing membranes virtually non-conductive in some experiments. The effects of both modulator activities could be completely reversed by the addition of pronase. Simple perfusion of the chamber did not reverse the effect of the modulator on VDAC. By controlling the gating of VDAC channels, the VDAC modulator could play an important role in regulating cellular metabolism.

Animals↗
PubMed1994

NADH regulates the gating of VDAC, the mitochondrial outer membrane channel.

Aerobic energy metabolism in cells involves the transfer of reducing equivalents from organic molecules to oxygen. NADH is important as a carrier of these reducing equivalents and as a feedback regulator of glycolysis. We report that micromolar quantities of NADH double the voltage dependence of the mitochondrial channel, VDAC, a critical pathway for the flux of metabolites between the cytoplasm and the mitochondrial spaces. In the presence of NADH, the opening and closing of this channel is more sensitive to changes in membrane potential and thus presumably better able to respond to changes in metabolic conditions. This effect was observed both on a human and two fungal forms of VDAC, indicating a highly conserved regulatory mechanism. NAD+ and other nucleotides tested failed to mimic the action of NADH. This ability of NADH to facilitate VDAC closure could be one mechanism by which glycolysis can suppress oxidative phosphorylation (Crabtree effect).

Amino Acid Sequence↗