Membrane vesicle-mediated transport

Clathrin-coated vesicles mediate transport from the Golgi apparatus to endosomes, and from the plasma membrane to endosomes. A multi-subunit protein, clath-rin, constitutes the major protein of this vesicle type (see Ch. 2). Clathrin is composed of three large and three small polypeptide chains, which assemble to form a triskelion (Fig. 9-2). Regulatory mechanisms control the assembly and formation of a convex, polyhexa-pentagonal basketlike structure by these triskelions [5], This structure is responsible for the formation of coated pits on the cytosolic face of plasma membranes. 

The fusion of membranes at several stages in the vesicle-mediated transport of materials between Golgi compartments requires a specific protein known as the N-ethylmaleimide-sensitive fusion protein... 

In two variations of this basic procedure, transport of VSV G protein is monitored by different techniques. Studies using both of these modern trafficking assays and Palade s early experiments all came to the same conclusion in mammalian cells vesicle-mediated transport of a protein molecule from its site of synthesis on the rough ER to its arrival at the plasma membrane takes from 30 to 60 minutes. 

In addition to passive diffusion, channels, and carrier proteins, a fourth mechanism of breaching biological membranes is provided by vesicle-mediated transport, which can also be used for drug delivery. Endocytosis refers to the uptake of cell surface constituents by the formation of vesicles from the plasma membrane. This process can be subclassified into phagocytosis, the engulfment of particles, and pinocytosis, the engulfment of fluid. 

Another important mechanism of vesicle-mediated transport is defined by the structural protein caveolin. Caveolins are a family of integral membrane proteins that self-associate and also specifically bind to cholesterol and glycosphingolipids in a relatively stable microdomain. These microdomains are an important... 

Vesicle-mediated transport Transport of a substance between cellular membranes with the substance either within or enclosed by a vesicle. 

Membrane fusion is an important process in many cellular events, such as fertilization, myoblast fusion, virus infection, secretion, and intracellular vesicle-mediated transport. Proteins are known to be essential for mediating membrane fusion in these events. However, the exact mechanisms for induction of membrane fusion by proteins are still unknown. Membrane fusion reactions consist of three steps close apposition of membranes, mixing of lipid molecules in the closely apposed membranes, and formation of new bilayers in a different direction. For elucidation of the mechanism, we have studied protein-induced membrane fusion using the protein clathrin and liposome membrane systems. 

The exocytotic release of neurotransmitters from synaptic vesicles underlies most information processing by the brain. Since classical neurotransmitters including monoamines, acetylcholine, GABA, and glutamate are synthesized in the cytoplasm, a mechanism is required for their accumulation in synaptic vesicles. Vesicular transporters are multitransmembrane domain proteins that mediate this process by coupling the movement of neurotransmitters to the proton electrochemical gradient across the vesicle membrane. 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

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.

Figure 11.1 Schematic representation of iron uptake mechanisms, (a) The transferrin-mediated pathway in animals involves receptor-mediated endocytosis of diferric transferrin (Tf), release of iron at the lower pH of the endocytic vesicle and recycling of apoTf. (b) The mechanism in H. influenzae involves extraction of iron from Tf at outer membrane receptors and transport to the inner membrane permease system by a periplasmic ferric binding protein (Fbp). From Baker, 1997. Reproduced by permission of Nature Publishing Group.

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]. 

Transport proteins (channels) for chloride and zinc Vacuolar proton pump Components of synaptic vesicles to mediate the chloride flux for glutamate uptake and zinc uptake in most synaptic vesicles. Zinc transporter is homologous to endosomal and plasma membrane zinc transporters chloride transporters remain to be identified. Protein complex of more than 12 subunits. Constitutes the largest component of synaptic vesicles and establishes... 

Loe, D.W., Almquist, K.C., Deeley, R.G. and Cole, S.P. (1996) Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. Journal of Biological Chemistry, 271, 9675—9682. 

Primarily to elucidate transporter localization and function, vesicles enriched in trophoblast apical or basolateral membranes have frequently been utilized. To give a few instances, they have been used to investigate P-gp-mediated transport, mechanisms of transport of cationic compounds, drug interactions with nutrient transport, and differences in amino acid transport in pathological conditions of the placenta [36, 40-42], Briefly, for preparation of microvillus membrane vesicles the cord, amniochorion and decidua are removed from placenta, and the tissue cut on the maternal side. The mince is stirred to loosen... 

F. Ushigome, N. Koyabu, S. Satoh, K. Tsukimori, H. Nakano, T. Nakamura, T. Uchiumi, M. Kuwano, H. Ohtani, and Y. Sawada. Kinetic analysis of P-glycoprotein-mediated transport by using normal human placental brush-border membrane vesicles. Pharm Res. 20 38-44 (2003). 

M. Takano, K. Inui, T. Okano, H. Satio, and R. Hori. Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles. Biochim Biophys Acta 773 113-124 (1984). 

Said, H.M., Redha, R., and Nylander, W., A carrier-mediated Na+ gradient-dependent transport for biotin in brush-border membrane vesicles, Am. ]. Physiol., 253, G631, 1987. 

It has been demonstrated that cis- (76) and frarcs-flupentixol (75) (see Fig. 5) inhibited the photo affinity labeling of P-gp by substrate analogues [173] Binding of several MDR modulators, among them TFP (5), to P-gp was shown by means of fluorescence quenching of the MIANS probe [174] or P-gp tryptophan fluorescence [175]. CPZ (9) is likely a P-gp substrate, as was shown in studies of its transport in membrane vesicles obtained from multidrug-resistant CCRF-CEM cells [176], and therefore it was used as a competitive inhibitor of drug transport mediated by P-gp [177]. 

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