Do Small Headgroups of Phosphatidylethanolamine and Phosphatidic Acid Lead to a Similar Folding Pattern of the K+ Channel?

Phospholipid headgroups act as major determinants in proper folding of oligomeric membrane proteins. The K+-channel KcsA is the most popular model protein among these complexes. The presence of zwitterionic nonbilayer lipid phosphatidylethanolamine (PE) is crucial for efficient tetramerization and stabilization of KcsA in a lipid bilayer. In this study, the influence of PE on KcsA folding properties was analyzed by tryptophan fluorescence and acrylamide quenching experiments and compared with the effect of anionic phosphatidic acid (PA). The preliminary studies suggest that the small size and hydrogen bonding capability of the PE headgroup influences KcsA folding via a mechanism quite similar to that observed for anionic PA

Protein-lipid interactions in assembly and function of Leader Peptidase

Cells are the entities of life and they at least consist of one aqueous compartment separated from the environment by a membrane. Lipids and proteins are important constituents of membranes and the interactions between these components are the subject of this thesis. The studies were performed using the model organism Escherichia coli and membrane fractions derived thereof, as well as with pure biochemical model systems comprising phospholipids and as a model membrane protein: Leader Peptidase. Leader Peptidase (Lep) is an integral membrane protein of E. coli and it catalyses the removal of signal peptides from translocated precursor proteins. In chapter 2 of this thesis, the number of Lep molecules per E. coli cell was determined using western blot techniques. Different strains were found to contain approximately 1000 Lep molecules per cell during exponential growth. In this thesis (Chapter 3) it is furthermore shown that when the membrane spanning segments of Lep are removed in vivo, the remaining catalytic domain can bind to inner and outer membranes of E. coli. The purified catalytic domain binds to inner membrane vesicles and vesicles composed of purified inner membrane lipids with comparable efficiency. It is shown that the interaction is caused by penetration of a part of the catalytic domain between the lipids. Penetration is mediated by phosphatidylethanolamine, the most abundant lipid in E. coli, and does not seem to depend on electrostatic interactions. A hydrophobic segment near the catalytically region of Lep is required for the interaction with membranes. The orientation of many membrane proteins is determined by the asymmetric distribution of positively charged amino acid residues in cytoplasmic and translocated loops. The positive-inside rule states that loops with large amounts of these residues tend to have cytoplasmic locations. In chapter 4, orientations of constructs derived from the Lep were found to depend on the anionic (negatively charged) phospholipid content of the membrane. Lowering the contents of anionic phospholipids facilitated membrane passage of positively charged loops. On the other hand, elevated contents of anionic phospholipids in the membrane rendered translocation more sensitive to positively charged residues. The results demonstrate that anionic lipids are determinants of membrane protein topology and suggest that interactions between negatively charged phospholipids and positively charged amino acid residues contribute to the orientation of membrane proteins. In chapter 5 a cell-free system based on a lysate and membrane vesicles from E. coli is used to study characteristics of the membrane integration reaction of Lep. Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease. It is concluded that integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids. Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro. In chapter 6 a structural models is proposed for the action of Lep, in this model the interactions of the various domains of Lep with precursor proteins in the phospholipid bilayer, are visualised. Chapter 6 also proposes models for the interactions between anionic lipids and membrane proteins in initial stages of insertion

S-Decyl-glutathione nonspecifically stimulates the ATPase activity of the nucleotide-binding domains of the human multidrug resistance-associated protein, MRP1 (ABCC1)

The human multidrug resistance-associated protein(MRP1) is an ATP-dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well-characterized multidrug transporter P-gp, MRP1 comprises two nucleotide-binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P-gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione-S -transferase (GST) or the maltose-binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST-fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent K (m) of 340 mum and a V (max) of 6.0 nmol P-I .mg(-1) .min(-1) , and a K (m) of 910 mum ATP and a V (max) of 7.5 nmol P-I .mg(-1) .min(-1) , respectively. Remarkably, S -decyl-glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2-fold in a concentration-dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S -decyl-glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations

Yeast Genes Controlling Responses to Topogenic Signals in a Model Transmembrane Protein

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2–3% by a strong charge difference signal. Identified pio mutants increased this to 9–12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca(2+) ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins