Effects of phospholipases C and D on ordering of channel proteins in the mitochondrial outer membrane (original) (raw)
Related papers
1986
The channel proteins in outer membranes of Neurospora crassa mitochondria spontaneously organize into periodic arrays when the membranes are dialyzed in the presence of soluble phospholipase A2. Electron microscopic images have been recorded at different electron doses from channel arrays in a variety of negative stains, as well as in vitreous ice. Fourier or correlation averages are formed from image fields containing a few hundred unit cells. These averages can be subsequently classified by correspondence analysis and summed to form representative averages over thousands of unit cells. In averages of negatively stained arrays, the stain-filled channel openings are bounded by smaller stain-excluding maxima. The projections of the channel openings are smaller and the subsidiary maxima are more pronounced for channel arrays contrasted with dilute (0.1%) uranyl acetate or aurothioglucose than for arrays embedded in 1% uranyl acetate. Projection images of unstained, ice-embedded membranes provide direct information about distribution of protein and lipid in the VDAC arrays. First experiments have yielded density maps with an apparent Fourier resolution of-1/(2 nm).
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1984
Antibodies were raised in rabbits against the outer membrane of Neurospora mitochondria. Antibodies were obtained that were specific for this membrane's major polypeptide (Mr 31 000) and its slower-migrating derivatives on SDS-polyacrylamide gels. These antibodies inhibited the insertion into phospholipid bilayers of voltage-dependent ion channels from detergent extracts of the mitochondrial outer membranes. The same antibodies bound preferentially to membranes containing crystalline surface arrays in outer mitochondrial membrane fractions. These results indicate that the 31 kDa polypeptide is a component both of the ion channels and of the membrane arrays, suggesting identity between the functional and structural entities.
Structure of the Channels in the Outer Mitochondrial Membrane
Biophysical Journal, 1986
The channel proteins in outer membranes of Neurospora crassa mitochondria spontaneously organize into periodic arrays when the membranes are dialyzed in the presence of soluble phospholipase A2. Electron microscopic images have been recorded at different electron doses from channel arrays in a variety of negative stains, as well as in vitreous ice. Fourier or correlation averages are formed from image fields containing a few hundred unit cells. These averages can be subsequently classified by correspondence analysis and summed to form representative averages over thousands of unit cells. In averages of negatively stained arrays, the stain-filled channel openings are bounded by smaller stain-excluding maxima. The projections of the channel openings are smaller and the subsidiary maxima are more pronounced for channel arrays contrasted with dilute (0.1%) uranyl acetate or aurothioglucose than for arrays embedded in 1% uranyl acetate. Projection images of unstained, ice-embedded membranes provide direct information about distribution of protein and lipid in the VDAC arrays. First experiments have yielded density maps with an apparent Fourier resolution of-1/(2 nm).
Proceedings of the National Academy of Sciences, 1983
The outer membrane of mitochondria contains proteins that form channels called VDAC (voltage-dependent anion-selective channels). Two independent lines of evidence suggest that these channels occur in specific complexes in the outer membrane of Neurospora mitochondria. Electron microscopic images of these outer membranes reveal polymorphic crystalline arrays of putative pores. These arrays can be shown to be interrelated by movement in the membrane plane of a particular rigid channel triplet or of regular aggregates of this triplet. Detergent extracts of the same outer membranes induce single- and multiple-step conductances in planar phospholipid membranes, with a marked preference for the insertion of triplets and multiples of triplets. The tendency of the mitochondrial channels to occur as extended arrays, apparently built up from triplets, may have important functional and evolutionary implications.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1989
Phospholilmse A 2 induces crystallization of the channel protein, VDAC (also called mitochondrial porin), in the outer membrane of Neurospora crassa mitochondria. The channel crystals formed in native membranes typically contain a few hundred unit cells. To increase the size of these membrane crystals for low-contrast electron microscopic imaging and diffraction slmlies, fusion of the isolated mitochondrial outer membranes was attempted before and after phospl~ipase treatment. Successful fusion of the untreated membranes was achieved by a procedure involving slow dehydration at acid pH. Single crystals of channels obtained by subsequent action of soluble phospholipase A 2 on fused mitoehondrlal outer membranes may contain several thousand unit cells.
Circular dichroism studies of the mitochondrial channel, VDAC, from Neurospora crassa
Biophysical Journal, 1996
The protein that forms the voltage-gated channel VDAC (or mitochondrial porin) has been purified from Neurospora crassa. At room temperature and pH 7, the circular dichroism (CD) spectrum of VDAC suspended in octyl /-glucoside is similar to those of bacterial porins, consistent with a high /3-sheet content. When VDAC is reconstituted into phospholipid liposomes at pH 7, a similar CD spectrum is obtained and the liposomes are rendered permeable to sucrose. Heating VDAC in octyl 8-glucoside or in liposomes results in thermal denaturation. The CD spectrum irreversibly changes to one consistent with total loss of /3-sheet content, and VDAC-containing liposomes irreversibly lose sucrose permeability. When VDAC is suspended at room temperature in octyl ,B-glucoside at pH <5 or in sodium dodecyl sulfate at pH 7, its CD spectrum is consistent with partial loss of /3-sheet content. The sucrose permeability of VDAC-containing liposomes is decreased at low pH and restored at pH 7. Similarly, the pH-dependent changes in the CD spectrum of VDAC suspended in octyl /-glucoside also are reversible. These results suggest that VDAC undergoes a reversible conformational change at low pH involving reduced /3-sheet content and loss of pore-forming activity.
Properties of channels in the mitochondrial outer membrane
Journal of Bioenergetics and Biomembranes, 1989
Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6 +_ 1 subunits.
Conformational change in the mitochondrial channel, VDAC, detected by electron cryo-microscopy
Biophysical Journal, 1993
Crystalline arrays of the voltage-dependent channel, VDAC, can be produced by treatment of Neurospora mitochondrial outer membranes with phospholipase A2. The membrane crystals undergo a lateral phase transition (lattice contraction) that can be induced by an amphipathic polyanion, which also reduces the channel's gating potential. Electron cryo-microscopy of frozen-hydrated crystals indicates that the mean projected diameters of the channels do not decrease with lattice contraction. Instead, contraction is associated with the disappearance of lateral protein "arms" that normally extend between the channels. A model is presented that explains the changes in channel packing and gating potential in terms of a conformational change involving the movement of a protein "arm" between the bilayer and the channel.
The Journal of Cell Biology, 1982
Light-membrane fractions obtained by hypoosmotic lysis of neurospora crassa mitochondria exhibit buoyant densities and marker-enzyme activities characteristic of outer mitochondrial membranes. SDS PAGE of these membrane fractions indicates that a polypeptide of M(r) 31,000 is the main protein component. Under negative-stain electron microscope examination many of the membranes in these fractions appear as large (0.5-1- mum diameter), collapsed vesicles. The surfaces of flattened, open (i.e., ripped) vesicles often exhibit extended two-dimensional arrays of subunits are arranged into hexagons within each parallelogram unit cell, 12.6x11.1 nm (lattice angle = 109 degrees).