Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

Extensive segregation of acidic phospholipids in membranes induced by protein kinase C and related proteins

Protein kinase C and two other proteins with molecular masses of 64 and 32 kDa, purified from bovine brain, constitute a type of protein that binds a large number of calcium ions in a phospholipid-dependent manner. This study suggested that these proteins also induced extensive clustering of acidic phospholipids in the membranes. Clustering of acidic phospholipids was detected by the self-quenching of a fluorescence probe that was attached to acidic phospholipids (phosphatidic acid or phosphatidylglycerol). Addition of these proteins to phospholipid vesicles containing 15% fluorescently labeled phosphatidic acid dispersed in neutral phosphatidylcholine resulted in extensive, rapid, and calcium-dependent quenching of the fluorescence signal. Fluorescence-quenching requirements coincided with protein-membrane binding characteristics. As expected, the addition of these proteins to phospholipid vesicles containing fluorescent phospholipids dispersed with large excess of acidic phospholipids produced only small fluorescence changes. In addition, association of these proteins with vesicles composed of 100% fluorescent phospholipids resulted in no fluorescence quenching. Protein binding to vesicles containing 5-50% fluorescent phospholipid showed different levels of fluorescence quenching that closely resemble the behavior expected for extensive segregation of the acidic phospholipids in the outer layer of the vesicles. Thus, the fluorescence quenching appeared to result from self-quenching of the fluorophores that become clustered upon protein-membrane binding. These results were consistent with protein-membrane binding that was maintained by calcium bridges between the proteins and acidic phospholipids in the membrane. Since each protein bound eight or more calcium ions in the presence of phospholipid, they may each induce clustering of a related number of acidic phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins that bind calcium in a phospholipid-dependent manner

Three proteins (Mr = 64K, 32K, and 22K) that bind to phospholipids in a calcium-dependent manner were purified from bovine brain. The calcium-binding properties of these proteins were investigated by equilibrium dialysis and by gel filtration chromatography. The 64- and 32-kDa proteins were found to have calcium- and phospholipid-binding properties strikingly similar to those of protein kinase C [Bazzi, M.D., & Nelsestuen, G.L. (1990) Biochemistry 29, 7624]. The free proteins bound limited divalent metal ion even at 200 microM calcium. However, they bound eight to nine calcium ions per protein in the presence of membranes containing acidic phospholipids. The calcium concentrations needed for protein-phospholipid binding were different for these two proteins and were strongly influenced by the phospholipid composition of the vesicles; vesicles of higher phosphatidylserine content required lower concentrations of calcium for protein-membrane association. These properties described a general type of calcium-interacting system where simultaneous interaction of all three components (protein, phospholipids, and calcium) is required. The free proteins may provide only partial coordinate bonds to each calcium ion, but complete calcium-binding sites could be generated at the protein-phospholipid interface. In contrast to the 64- and 32-kDa proteins, the 22-kDa protein bound similar amounts of calcium (two to three ions/protein) in the presence or the absence of phospholipids. The 22-kDa protein had the lowest affinity for phospholipid and the highest affinity for calcium of the three proteins tested. Thus, calcium-dependent phospholipid-binding proteins consist of several types. For example, the 64- and 32-kDa proteins appear to be quite abundant and may even function as a calcium buffer to modulate signaling events.     

High-affinity binding of NADPH to camel lens zeta-crystallin

Fluorescence spectrum of camel lens zeta-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including alpha-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with zeta-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and zeta-crystallin with an apparent Km<0.45 microM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of zeta-crystallin, also was able to quench the fluorescence of zeta-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that zeta-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in zeta-crystallin.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

Identifying QTLs for fire-blight resistance via a European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) genetic linkage map

The existence of different levels of susceptibility to fire blight (Erwinia amylovora) in European pear (Pyrus communis L.) cultivars suggests that it is possible to identify QTLs related to resistance in pear germplasm. Given the polygenic nature of this trait, we designed two genetic maps of the parental lines 'Passe Crassane' (susceptible) and 'Harrow Sweet' (resistant) using SSRs, MFLPs, AFLPs, RGAs and AFLP-RGAs markers. RGA-related markers should theoretically map in chromosome regions coding for resistance genes. The 'Passe Crassane' map includes 155 loci, for a total length of 912 cM organised in 18 linkage groups, and the 'Harrow Sweet' map 156 loci, for a total length of 930 cM divided in 19 linkage groups; both maps have a good genome coverage when compared to the more detailed apple maps. Four putative QTLs related to fire blight resistance were identified in the map. A suite of molecular markers, including two AFLP-RGAs, capable of defining resistant and susceptible haplotypes in the analysed population was developed.