Mini-Reviews June 23 The tale of tail-anchored proteins : coming from the cytosol and looking for a membrane Nica Borgese , Nica Borgese. This Site. Google Scholar. Sara Colombo , Sara Colombo. Emanuela Pedrazzini Emanuela Pedrazzini.
Author and Article Information. Nica Borgese. Sara Colombo. Emanuela Pedrazzini. Fax: E-mail: n. Received: April 03 Revision Received: May 14 Accepted: May 14 Online Issn: The Rockefeller University Press. J Cell Biol 6 : — Article history Received:. Revision Received:. Cite Icon Cite. Abell, B. Jung, J. Oliver, B. Knight, J. Tyedmers, R. Zimmermann, and S.
Search ADS. Blobel, G. Borgese, N. Gazzoni, M. Barberi, S. Colombo, and E. Bulbarelli, A. Sprocati, M. Barberi, E. Pedrazzini, and N. Chen, Y. Colbeau, A.
Nachbaur, and P. Cory, S. Dailey, H. De Silvestris, M. D'Arrigo, and N. Elgersma, Y. Kwast, M. Snyder, B. Distel, S. Subramani, and H. He, H. Lam, T. McCormick, and C. Honsho, M. Mitoma, and A. Isenmann, S. Khew-Goodall, J. Gamble, M. Vadas, and B. Toikkanen, C. Ehnholm, H. Jarvis, P. Joglekar, A. Xu, D.
Rigotti, R. Fairman, and J. Kaufmann, T. Schlipf, J. Sanz, K. Neubert, R. Stein, and C. Kim, P. Janiak-Spens, W. Trimble, B. Leber, and D. Hollerbach, W. Kuroda, R. Enzymes are highly specific to their substrates. They bind these substrates at complementary areas on their surfaces, providing a snug fit that many scientists compare to a lock and key.
Enzymes work by binding one or more substrates, bringing them together so that a reaction can take place, and releasing them once the reaction is complete. In particular, when substrate binding occurs, enzymes undergo a conformational shift that orients or strains the substrates so that they are more reactive Figure 3. The name of an enzyme usually refers to the type of biochemical reaction it catalyzes. For example, proteases break down proteins, and dehydrogenases oxidize a substrate by removing hydrogen atoms.
As a general rule, the "-ase" suffix identifies a protein as an enzyme, whereas the first part of an enzyme's name refers to the reaction that it catalyzes.
Figure 3: Enzymes and activation energy Enzymes lower the activation energy necessary to transform a reactant into a product.
On the left is a reaction that is not catalyzed by an enzyme red , and on the right is one that is green. In the enzyme-catalyzed reaction, the enzyme binds to the reactant and facilitates its transformation into a product.
Consequently, the enzyme-catalyzed reaction pathway has a smaller energy barrier activation energy to overcome before the reaction can proceed. The proteins in the plasma membrane typically help the cell interact with its environment. For example, plasma membrane proteins carry out functions as diverse as ferrying nutrients across the plasma membrane, receiving chemical signals from outside the cell, translating chemical signals into intracellular action, and sometimes anchoring the cell in a particular location Figure 4.
Figure 4: Examples of the action of transmembrane proteins Transporters carry a molecule such as glucose from one side of the plasma membrane to the other. Receptors can bind an extracellular molecule triangle , and this activates an intracellular process. Enzymes in the membrane can do the same thing they do in the cytoplasm of a cell: transform a molecule into another form.
Anchor proteins can physically link intracellular structures with extracellular structures. Figure Detail. The overall surfaces of membrane proteins are mosaics, with patches of hydrophobic amino acids where the proteins contact lipids in the membrane bilayer and patches of hydrophilic amino acids on the surfaces that extend into the water-based cytoplasm. Many proteins can move within the plasma membrane through a process called membrane diffusion. This concept of membrane-bound proteins that can travel within the membrane is called the fluid-mosaic model of the cell membrane.
The portions of membrane proteins that extend beyond the lipid bilayer into the extracellular environment are also hydrophilic and are frequently modified by the addition of sugar molecules. Other proteins are associated with the membrane but not inserted into it. They are sometimes anchored to lipids in the membrane or bound to other membrane proteins Figure 5. GPI-defective cells from patients with paroxysmal nocturnal hemoglobinuria PNH are relatively resistant to apoptosis induction.
Using retroviral PIG-A gene transfer along with the transfer of a vector control, the authors obtained two genetically identical cell lines, distinguished only by expression of the PIG-A gene and, thus, their ability to produce GPI. Cell proliferation and survival were not affected by this difference. Apoptotic stimuli such as serum starvation and camptothecin exposure elicited similar responses. Parasite glycosylphosphatidylinositol GPI is an important toxin in malaria disease, and people living in malaria-endemic regions often produce high levels of anti-GPI antibodies.
The natural anti-GPI antibody response needs to be understood to aid the design of an efficient carbohydrate-based antitoxin vaccine. The authors present a versatile approach based on a synthetic GPI glycan array to correlate anti-GPI antibody levels and protection from severe malaria.
Schofield, Louis; Hewitt, Michael C. Fatalities are thought to result in part from pathol. Glycosylphosphatidylinositol GPI originating from the parasite has the properties predicted of a toxin; however, a requirement for toxins in general and GPI in particular in malarial pathogenesis and fatality remains unproven. As anti-toxic vaccines can be highly effective public health tools, the authors sought to det. The P. Recipients were substantially protected against malarial acidosis, pulmonary edema, cerebral syndrome, and fatality.
Anti-GPI antibodies neutralized pro-inflammatory activity by P. Thus, GPI is a pro-inflammatory endotoxin of parasitic origin, and several disease parameters in malarious mice are toxin-dependent.
GPI may contribute to pathogenesis and fatalities in humans. Synthetic GPI is therefore a prototype carbohydrate anti-toxic vaccine against malaria. Munksgaard International Publishers Ltd. In recent years, the technol. We have prepd. The ability to form PrPSc in transgenic mice is retained by a residue "mini-prion" PrP , with the deletions and A single reversed-phase purifn.
With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP was indistinguishable from its recombinant analog rPrP Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine Bo PrP , were successfully prepd. To mimic the glycosylphosphatidyl inositol GPI anchor and target sPrP to cholesterol-rich domains on the cell surface, where the conversion of PrPC is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences.
The chem. Conversion of cellular prion protein PrPC into the pathol. However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amt.
PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP rPrPPalm via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells.
These latter results enable expts. Paulick, Margot G. National Academy of Sciences. The glycosylphosphatidylinositol GPI anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biol. The authors previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chem.
Here the authors investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor.
The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion.
Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biol. Protein Pept. Bentham Science Publishers Ltd. In living cells, membrane proteins are essential to signal transduction, nutrient use, and energy exchange between the cell and environment. Due to challenges in protein expression, purifn. This review describes recent advances in soln.
NMR allowing the study of a select set of peripheral and integral membrane proteins. These structural studies are possible due to solubilization of the proteins in membrane-mimetic constructs such as detergent micelles and bicelles. These examples illustrate the unique role soln. NMR spectroscopy plays in structural biol.
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Journal of Inherited Metabolic Disease , 44 3 , ChemPhysChem , 22 8 , The following figure 3 summarizes membrane protein functions for easy to understand. Expression of Membrane Proteins. Despite significant and considerable recent improvements, the expression of functionally folded membrane proteins in sufficient amounts for functional and structural studies is still a challenging task.
Compared with soluble cytoplasmic proteins, a variety of difficulties in expression of membrane proteins. Membrane proteins are not just released into the cytosol but must rather be targeted and translocated to their final destinations in membranes. In particular in eukaryotic cells, there is need a more complicated biological process requires sophisticated recognition and sorting mechanisms.
Copy number and capacity of prokaryotic and eukaryotic expression systems are limited because of translocation machineries, and translocation can be selective only for distinct groups of membrane proteins. For structural and functional studies or other purposes, it is have to extract membrane protein from cellular after expression, followed by transfer into artificial and defined hydrophobic environments like micelles or liposomes.
In this procedure, it is highly critical as membranes have to be disintegrated by relatively harsh detergents which can result in conformational aberrations or in unfolding of membrane proteins. Core factors that determine the yield, integrity, activity and stability of synthesized membrane proteins mainly are the availability of highly processive transcription and translation machineries, suitable folding environments, the lipid composition of cellular membranes, the presence of efficient targeting systems and appropriate pathways for posttranslational modifications PTMs.
However, the efficiency and workload of the individual expression systems are quite different, and preparative amounts of membrane proteins are often only obtained with bacterial, yeast or cell-free systems Fig.
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