The Outer and Inner Membranes

Our knowledge of the organization and functions of the outer and inner membranes has been considerably enlarged by studies on the effects of chemical treatments and biosynthetic blocks. 

It is now evident that divalent cations play an important role in the interaction of the lipopolysaccharide, phospholipid, and protein components of the outer membrane. When intact cells are treated with EDTA, as much as 90% of the LPS, 5-10% of the protein, and 5% of the phospholipid of the membrane are released. There is also some evidence that the remaining LPS represents a fraction which is newly synthesized 

Before describing these results, it is necessary to consider the structure of a normal cell. A freeze-etched surface of untreated cells is shown in Fig. 2. A rather smooth outermost surface is observed after etching times of 1-3 min. This surface is the layer to which bacteriophages adsorb. At higher 

The ultrastructure of the plasma membrane was also affected in the treated cells. The intramembranous particles appeared coarser and were, in general, more randomly distributed, so that (after cooling) the particle-free areas seen in the hydrophobic region of the inner membrane of control cells were found only occasionally, and even then they were relatively ill defined. These changes were less marked if the cells were not treated with CaCU prior to EDTA. While it was not possible to reverse the structural alterations by addition of CaCl2, a short period of growth of the cells after dilution into fresh medium abolished the changes and reestablished the sur- 

It was noted earlier that the bilaminar appearance of the outer membrane seen in ultrathin sections is most likely associated with the core and lipid A regions of the LPS. Mutants with altered polysaccharides do not show marked differences in the membrane profile, with the exception of a possible decrease in the thickness of the bilayer. Therefore, chemical-structural changes in the LPS component must be probed with more sensitive but indirect methods, such as measurements of the separation space between adjacent cells or labeling with ferritin-tagged antibodies. It is also possible that mutations affecting LPS could have secondary effects on the distribution and quantity of other components which could mask structural changes due to the altered LPS. 

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The mechanism of action of defensins is largely unknown. Incubation with defensins results in the formation of voltage-regulated ion channels that permeabilise the outer and inner membranes of metabolically-active E. coli. Because the target bacteria must be metabolically active for defensins to exert their effects, it may be that the transmembrane electromotive force is involved in the mechanism of action. 

Many bacteria secrete a wide range of proteins including pathogenic factors such as toxins. They must pass through both the outer and inner membranes. There are various mechanisms for protein secretion. Among them, three pathways are conserved in many species of gram-negative bacteria (Salmond and Reeves, 1993 Nunn, 1999). 

Figure 18-2 (A) Schematic diagram of mitochondrial structure. (B) Model showing organization of particles in mitochondrial membranes revealed by freeze-fracture electron microscopy. The characteristic structural features seen in the four half-membrane faces (EF and PF) that arise as a result of fracturing of the outer and inner membranes are shown. The four smooth membrane surfaces (ES and PS) are revealed by etching. From Packer.8...

Fig. 12.5. Biogenesis and assembly of cytochrome 6-c, complex in the inner mitochondrial membrane. Cytochrome fc-Cj complex contains at least five different subunits COREI (corl), COREII (corll), nonheme iron protein (Fe-S), cytochrome c, (cyt Cj), and cytochrome b (cyt b). Cytochrome f> is a mitochondrial gene product and is probably assembled into the inner membrane (IM) via vectorial translation by mitochondrial ribosomes. The other subunits are synthesized on cytoplasmic ribosomes as larger precursors. The precursors, perhaps in association with a cytoplasmic factor , are attached to receptors on the mitochondrial outer membrane (OM). The complex laterally diffuses to the junctions of the outer and inner membranes, and with the help of a hypothetical translocator the precursors are imported across the membrane. Pre-Corl, pre-Corll, and the pre-nonheme iron protein cross the two membranes, whereas cytochrome c, becomes anchored to the outer face of the inner membrane, facing the intermembrane space (IMS). Cytochrome b is assembled inside the inner membrane, and the nonheme iron protein and Corl and Corll are assembled into the matrix side of the inner membrane. The N-terminal extensions are removed by a soluble matrix protease. The N-terminal extension of cytochrome c, is removed in two steps the first is catalyzed by the matrix protease and the second probably by a protease located on the outer face of the inner membrane.

As you hopefully recall, the parts of a chloroplast include the outer and inner membranes, intermembrane space, stroma, and thylakoids stacked in grana. The chlorophyll is built into the membranes of the thylakoids. 

Mitochondrial and chloroplast proteins typically pass through the outer and inner membranes to enter the matrix or stromal space, respectively. Other proteins are sorted to other subcompartments of these organelles by additional sorting steps. Nuclear proteins enter through visible nuclear pores by processes discussed in Chapter 12. 

Microscopic studies of stable translocation Intermediates show that they accumulate at sites where the inner and outer mitochondrial membranes are close together, evidence that precursor proteins enter only at such sites (Figure 16-27c). The distance from the cytosolic face of the outer membrane to the matrix face of the inner membrane at these contact sites is consistent with the length of an unfolded spacer sequence required for formation of a stable translocation intermediate. Moreover, stable translocation Intermediates can be chemically cross-linked to the protein subunits that comprise the translocation channels of both the outer and inner membranes. This finding demonstrates that imported proteins can simultaneously engage channels in both the outer and inner mitochondrial membrane, as depicted in Figure... 

Cytosolic chaperones maintain the precursors of mitochondrial and chloroplast proteins in an unfolded state. Only unfolded proteins can be Imported into the organelles. Translocation occurs at sites where the outer and inner membranes of the organelles are close together. 

Proteins destined to the mitochondrial matrix bind to receptors on the outer mitochondrial membrane, and then are transferred to the general import pore (Tom40) in the outer membrane. Translocation occurs concurrently through the outer and inner membranes, driven by the proton-motive force across the inner membrane and ATP hydrolysis by the Hsc70 ATPase in the matrix (see Figure 16-26). 

Fig. 8. Interorganelle transport of PC and PE from the ER and plasma membranes. The structure S> represents the diacylglycerol portion of the phospholipid, and represents the fluorescent diacylglycerol of phospholipids. PCho and PEtn are the abbreviations for phosphocholine and phosphoethanolamine, respectively. OM and IM are abbreviations for the outer and inner membranes of the mitochondria. The for PC transport from the perinuclear region of the cell to the plasma membrane is shown in brackets and estimated to be 20 min. The majority of NBD-PCho transport from the plasma membrane occurs by a clathrin-dependent mechanism.

Johnson, D. Lardy, H. (1967) Isolation of liver or kidney mitochondria. Methods Emymol 10, 94-96. Parsons, D.F., Williams, G.R. Chance, B. (1966) Characteristics of isolated and purified preparations of the outer and inner membranes of mitochondria. Ann. New. York. Acad. Sci. 137, 643-666. 

Zq + <5 )i + ( Zq - 1)x + a 0 Here the dimensionless variable x characterises proton concentration within the membrane, y reflects proton permeability of the outer and inner membrane surfaces, all other symbols standing for some adjustabl parameters ... 

Basically, all mitochondria consist of two membranes which surround and enclose an inner compartment containing the mitochondrial matrix. The outer membrane is usually smooth in surface contour, whereas the inner membrane is folded into a series of lamellae, the cristae. The regions of the inner membrane between the cristae are designated collectively as the inner boundary membrane. The space between the outer and inner membranes, known as the inter membrane space, is continuous with the space bounded by the membranes of the cristae. Figure 1 shows a schematic drawing of a mitochondrion. 

Yolk formation within the mitochondrion begins with the appearance of a small granule between the outer and inner membranes or between the two layers of a crista. As the yolk granule enlarges, the space between the cristae and the matrix is much reduced or disappears completely. However, as Massover (1971) has pointed out, it seems unlikely that the cristae are simply pushed out of the way by the enlarging yolk body since empty regions around yolk granules are frequently seen. Moreover, similar dilations of intracristal space have been noted in various other mitochondria not involved in yolk formation (Wischnitzer, 1967 Massover, 1971). 

Electron microscopy of M. shows a characteristic internal structure. There are 2 concentric membranes, each 5-7 nm thick (see Biomembranes). Between the outer and inner membranes lies the intermembrane space (also called external matrix or outer mitochondrial space) (Figs. 1 2). These 2 membranes have different submicroscopic structures, their biogenesis is different, and they are functionally distinct. The outer membrane can be removed by osmotic rupture. 

Isolated Wistar rat liver mitochondria accumulated Fe(III) partly by an energy-dependent and partly by an energy independent mechanism (Romslo and Flatmark 1973). When the iron-loaded mitochondria were disrupted mechanically and the mitochondrial subfractions isolated by density gradient centrifugation, the iron accumulated by the energy-dependent mechanism was recovered mainly in the soluble matrix and intermembrane space (approx. 50 % of the total activity) and the inner membrane (approx. 30% Romslo and Flat-mark 1974). On the other hand, most of the energy-independent iron accumulation was confined to the outer and inner membranes (approx. 35 % of the total activity in each). 

Various relationships between the inner membrane in the mitochondrial cavity and the outer membrane delimiting the mitochondria have been described. Inside the mitochondria, the inner membrane may form independent septa or lamellae in a cavity that remains continuous. Another possibility is that the outer and inner membranes are in fact intimately connected. In that case, the septa may either form infolds of the three layers of the mitochondrial membrane or may just be projections of the inner electron-dense layer of the outer mitochondrial membrane. It becomes important to determine whether these projections end abruptly at some distance from the outer membrane or if they extend throughout the entire width of the mitochondrial cavity to form complete septa separating the main mitochondrial cavity into numerous smaller cavities. 

Parsons, D. F., and Yano, Y. (1967). The cholesterol content of the outer and inner membranes of guinea-pig fiver mitochondria. Biochim. Biophys. Acta 135, 362-364. 

The phenol-soluble envelope fraction of Escherichia coli has been shown to consist of at least three distinct glycoproteins one of them has an appreciable rate of turnover and is thought to be involved in the transfer of carbohydrates in the envelope. Analysis of the outer- and inner-membrane glycoproteins showed that they are not identical. The principal envelope protein of Halobacterium salinarium is also a glycoprotein of molecular weight 1.94 x 10 . ... 

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