Cytoplasmic membrane vesicles

To study transport in the absence of complicating metabolic processes, it often is advantageous to work with isolated membrane vesicles rather than with whole cells. Cytoplasmic membrane vesicles can be obtained from either eukaryotic or bacterial cells after homogenization or osmotic lysis. Transport proteins that have been solubilized with detergents also can be reincorporated into synthetic phospholipid vesicles (fig. 17.27). 

Most results on calcium transport have been obtained using cytoplasmic membrane vesicles, which may be prepared in inside-out or right-side-out configurations. Inside-out vesicles may be obtained by the disruption of E. coli cells in a French press. These then accumulate Ca2+ in an energy-dependent fashion, provided ATP or an oxidizable substrate is available. Addition of phosphate enhances the uptake of calcium as calcium phosphate is precipitated inside the tell, thus accounting for the lack of exchangeability of the calcium.186... 

Yu. J.L.. Crinius, L.. and Hooper, D.C. (2002) NorA functions as a multidrug efflux protein in both cytoplasmic membrane vesicles and reconstituted proteoliposomes. Journal of Bacteriology, 184 (5). 1370-1377. 

Fig. 12. Electron micrograph of thin section through cytoplasmic membrane vesicles from Bacillus suhlilis.

Studies of the lactose transport activity of cytoplasmic membrane vesicles from E. coli have demonstrated the functional symmetry of the jS-D-galacto-sidase carrier in the organism. ... 

Shechter, E., Letellier, L., and Gulik-Krzywicki, 1974, Relations between structure and function in cytoplasmic membrane vesicles isolated from an E. coli fatty acid auxo-troph, Eur. J. Biochem. 49 61. 

Even though dynein, kinesin, and myosin serve similar ATPase-dependent chemomechanical functions and have structural similarities, they do not appear to be related to each other in molecular terms. Their similarity lies in the overall shape of the molecule, which is composed of a pair of globular heads that bind microtubules and a fan-shaped tail piece (not present in myosin) that is suspected to carry the attachment site for membranous vesicles and other cytoplasmic components transported by MT. The cytoplasmic and axonemal dyneins are similar in structure (Hirokawa et al., 1989 Holzbaur and Vallee, 1994). Current studies on mutant phenotypes are likely to lead to a better understanding of the cellular roles of molecular motor proteins and their mechanisms of action (Endow and Titus, 1992). 

Histopathological features are dominated by the large number of centrally-placed muscle nuclei, sometimes affecting more than 90% of muscle fibers. The nuclei form long chains in the middle of the fiber and are surrounded by cytoplasm, which contains mitochondria and membranous vesicles, but no myofibrils. This morphological appearance has prompted comparison with myotubes, and in fact centronuclear myopathies are sometimes referred to as myotubular myopathies. This is a misnomer, however, since although the affected fibers retain some of the structural features of myotubes, and maturational arrest may play a role in their formation, the vast majority of such fibers are fully differentiated histochemically into either type 1 or type 2. 

The mechanisms involved in the establishment of lipid asymmetry are not well understood. The enzymes involved in the synthesis of phospholipids are located on the cytoplasmic side of microsomal membrane vesicles. Translocases (flippases) exist that transfer certain phospholipids (eg, phosphatidylcholine) from the inner to the outer leaflet. Specific proteins that preferentially bind individual phospholipids also appear to be... 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Figure 1 General pathways through which molecules can actively or passively cross a monolayer of cells. (A) Endocytosis of solutes and fusion of the membrane vesicle with the opposite plasma membrane in an active process called transcytosis. (B) Similar to A, but the solute associates with the membrane via specific (e.g., receptor) or nonspecific (e.g., charge) interactions. (C) Passive diffusion between the cells through the paracellular space. (C, C") Passive diffusion (C ) through the cell membranes and cytoplasm or (C") via partitioning into and lateral diffusion within the cell membrane. (D) Active or carrier-mediated transport of an otherwise poorly membrane permeable solute into and/or out of a cellular barrier.

Lipids are transported between membranes. As indicated above, lipids are often biosynthesized in one intracellular membrane and must be transported to other intracellular compartments for membrane biogenesis. Because lipids are insoluble in water, special mechanisms must exist for the inter- and intracellular transport of membrane lipids. Vesicular trafficking, cytoplasmic transfer-exchange proteins and direct transfer across membrane contacts can transport lipids from one membrane to another. The best understood of such mechanisms is vesicular transport, wherein the lipid molecules are sorted into membrane vesicles that bud out from the donor membrane and travel to and then fuse with the recipient membrane. The well characterized transport of plasma cholesterol into cells via receptor-mediated endocytosis is a useful model of this type of lipid transport. [9, 20]. A brain specific transporter for cholesterol has been identified (see Chapter 5). It is believed that transport of cholesterol from the endoplasmic reticulum to other membranes and of glycolipids from the Golgi bodies to the plasma membrane is mediated by similar mechanisms. The transport of phosphoglycerides is less clearly understood. Recent evidence suggests that net phospholipid movement between subcellular membranes may occur via specialized zones of apposition, as characterized for transfer of PtdSer between mitochondria and the endoplasmic reticulum [21]. 

Vesicles containing small numbers of separate rosettes have been found in the cytoplasm of Micrasterias during primary growth,367 suggesting that microfibril-synthesizing units are assembled in cytoplasmic membranes, and are incorporated into the plasma membrane by similar mechanisms during primary- and secondary-wall formation. 

After the binding, the virus may use various means of penetration (a) direct penetration, via the translocation of the entire virus through the cytoplasmic membrane (b) endocytosis, which is mediated by receptors, resulting in the formation of intercytoplasmic vesicles containing many viral particles (c) direct fusion of the viral envelope with the cytoplasmic membrane. 

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