Plasma membrane of yeast

Other ATP-dependent proton pumps are present in the plasma membranes of yeast and other fungi274b and also in the acid-secreting parietal cells of the stomach (Fig. 18-17). The H+-ATPase of Neurospora pumps H+ from the cytoplasm without a counterion. 

The proteins of membranes often include enzymes, e.g. adenosine triphosphatase which occurs in the plasma membrane of yeast where it assists uptake of aminoacids (Post et aL i960) and permeases (Section 2.1) are commonly found in membranes. It is logical to suppose that the protruding parts of the membrane proteins are made mainly of aminoacids with polar side-chains, whereas the embedded parts are rich in aminoacids with non-polar side-chains. 

Marx, U., Polakowski, T., Pomorski, T., Lang, C., Nelson, H., Nelson, N. and Herrmann, A., 1999, Rapid transbUayer movement of florescent phosphohpid analogs in the plasma membrane of endocytosis-deficient yeast cehs does not require the Drs2 protein. Eur. J. Biochem. Hai 254-263. 

An H+ electrochemical gradient (ApH+) provides the energy required for active transport of all classical neurotransmitters into synaptic vesicles. The Mg2+-dependent vacuolar-type H+-ATPase (V-ATPase) that produces this gradient resides on internal membranes of the secretory pathway, in particular endosomes and lysosomes (vacuole in yeast) as well as secretory vesicles (Figure 3). In terms of both structure and function, this pump resembles the F-type ATPases of bacteria, mitochondria and chloroplasts, and differs from the P-type ATPases expressed at the plasma membrane of mammalian cells (e.g., the Na+/K+-, gastric H+/K+-and muscle Ca2+-ATPases) (Forgac, 1989 Nelson, 1992). The vacuolar and F0F1... 

Copper uptake across the gastrointestinal tract is poorly understood — most probably utihsing the divalent cation transporter DMTl. At the cellular level, Cu is imported across the plasma membrane of mammalian cells as Cu, by members of the CTR family. The CTR family of proteins have been found in yeast and plants, as we saw, but also in humans and other mammals. They contain several methionine-rich motifs at their N-terminus, and conserved cysteine and histidine residues at their C-terminus. Unusually, CTR proteins can mediate the uptake of platinum anticancer drugs into mammalian cells (see Chapter 22). 

The question of how DnaJ proteins interact with substrates and mediate their transfer onto Hsp70 partner proteins is not answered for any of the three classes of DnaJ proteins. Some DnaJ homologs have broad substrate specificity, such as E. coli DnaJ and yeast Ydjl, while others have more restricted substrate spectra. In particular the DnaJ proteins of class III may either bind a restricted number of substrates, such as the clathrin-specific auxilin or the kinesin light chain, or they may not bind substrates themselves but rather are positioned in close proximity to substrates. The latter seems to be the case for Dj 1A in the plasma membrane of E. coli (Clarke et al, 1997 Kelley and Georgopoulos, 1997a), Sec63 at the translocation pore in the ER (Corsi and Schekman, 1996 Rapoport et al., 1996), and cysteine string proteins on the surface of neurosecretory vesicles (Buchner and Bundersen, 1997). 

The mechanism of this transport is presently unknown, but the results are consistent with a soluble carrier mechanism such as PC transfer protein, or transport at zones of apposition between membranes that facilitate rapid intermembrane transfer. Work by Pichler and colleagues has identified a subfraction of the ER that associates closely with the plasma membrane in yeast (H. Pichler, 2001). Future studies examining the effects of agents or mutations that disrupt these intracellular membrane associations will be critical for determining their role in lipid traffic. 

Eukaryotic as well as most prokaryotic membranes are bllayers of phospholipid and protein. Each monolayer, which is about 2.1 nm thick (19,20), is believed to contain the sterol in a nonhomogeneous distribution, and at least in some cases sterol can move between the monolayers. This process is called "flip-flop". Sterol has been found both in the mitochondria (21) and the plasma membrane of cerevlslae (13.18), and the ability to support the growth of anaerobic yeast presumably Is associated with Its membranous function. 

At higher ethanol concentrations the intracellular alcohol interferes with membrane organization, increasing its fluidity and permeability to ions and small metabolites and inhibiting transport of nutrients. Especially Ca and Mg ions are able to increase the plasma membrane stability. It has been demonstrated that incorporation of unsaturated fatty acids and/or sterol(s) as well as proteolipids into cellular membrane of yeasts helps to alleviate ethanol tolerance. For the synthesis of the unsaturated fatty acids the presence of traces of oxygen under fermentation conditions is required. Further to Ca and Mg ions, other trace elements such as Co, Cu, Mn and Zn " and vitamins, e.g. pantothenate, thiamine, riboflavin, nicotinic acid, pyridoxine, biotin, folic acid and inositol, are essential for the growth and ethanol production by yeasts. 

In prokaryotic cells fatty acid synthesis occurs in the cytosolic compartment. However, it has been observed that ACP in E. coli appears to be somewhat loosely associated with the inner face of the plasma membrane of the cell (van den Bosch et al., 1970). Nevertheless, all the activities associated with the synthesis of palmitic acid from acetyl-CoA can be readily separated and assigned to individual proteins which have been purified and their molecular and kinetic characteristics examined in considerable detail (Vagelos, 1974). In yeast and animal cells, the fatty acid synthetase responsible for the formation of palmitic acid is always associated with the cytosolic compartment as a dimer of a polyfunctional polypeptide (ibid.). 

Therefore, if the recipient cell does not express the specific lipids required by the receptor (which may concern the acyl chain content of sphingolipids), or an adequate cholesterol-sphingolipid balance, the transfection experiment may lead to an abxmdant expression of a totally inactive receptor. In 2003, Opekarova and Tanner published a list of more than 30 membrane proteins whose activity is specifically affected by lipids. The list covered a broad range of proteins expressed by various bacteria, yeasts, insect, and mammalian cells. The problem is particularly acute when mammalian receptors or transporters are expressed in bacteria. For instance, the failure to express fxmctional serotonin transporters in E. coli has been attributed to the lack of cholesterol in bacteria. Moreover, the recovery of fully active neurotensin and adenosine receptors in transfected bacteria required the presence of choles-teryl hemisuccinate (a cholesterol derivative) during solubilization. Paradoxical results have also been obtained for some proteins whose activity requires cholesterol but can be fxmctionally expressed in bacterial hosts. In this case, one can exclude a direct interaction of cholesterol with the protein but rather consider a more general effect of the sterol on membrane properties. As a matter of fact, we are just at the beginning of our comprehension of the complex molecular ballet that involves bofh lipid and protein actors in the plasma membrane of excitable cells. 

FIGURE 10.18 A model for the structure of the a-factor transport protein in the yeast plasma membrane. Gene duplication has yielded a protein with two identical halves, each half containing six transmembrane helical segments and an ATP-binding site. Like the yeast a-factor transporter, the multidrug transporter is postulated to have 12 transmembrane helices and 2 ATP-binding sites. 

In Saccharomyces cerevisiae, as in most eukaryotic cells, the plasma membrane is not freely permeable to nitrogenous compounds such as amino acids. Therefore, the first step in their utilization is their catalyzed transport across the plasma membrane. Most of the transported amino acids are accumulated inside the yeast cells against a concentration gradient. When amino acids are to be used as a general source of nitrogen, this concentration is crucial because most enzymes which catalyze the first step of catabolic pathways have a low affinity for their substrates. 

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