Using 31P NMR and freeze‐fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non‐bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.
Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated rapidly in cells stimulated by various extracellular signals. With stimulation of quiescent cells by growth factors, activated p42/p44 MAP kinases rapidly translocate to the nucleus, where they induce immediate early gene transcription. The MAP kinase signal transduction pathway represents an important mechanism by which growth factors regulate cellular events such as cell cycle progression or cell growth. In the present study, p42MAPK (ERK2) was studied during the ongoing cell cycle of Chinese hamster ovary cells synchronized by mitotic shake-off. We show that protein expression of p42MAPK increased in mid-G1 and that MAP kinase is phosphorylated during G1, as visualized by a gel-mobility shift and by the use of phosphospecific antibodies. This phosphorylation appeared to occur in the cytoplasm rather than at the plasma-membrane. In addition, phosphorylated p42MAPK was found to translocate to the nucleus during late/mid-G1. Treatment of cells with MEK inhibitor PD098059 prevented the phosphorylation and nuclear translocation of MAP kinase and DNA synthesis. Thus, nuclear translocation of p42MAPK is not restricted to the G0/G1 transition but occurs in every cell cycle and seems to be required for cell cycle progression.
Phosphatidylethanolamine (PE) is a nonbilayer-preferring and fusogenic phospholipid. It is kept in the bilayer configuration by interaction with other phospholipids in biologic membranes. However, reorganization of the membrane phospholipids could lead to expression of the nonbilayer nature of PE and induce bilayer instability. During ischemia a transbilayer reorganization of sarcolemmal PE is observed, and results have been published that suggest a lateral phase separation in the inner sarcolemmal leaflet phospholipids. These reorganizations and the subsequent expression of the nonbilayer behavior of PE are proposed to form the basis for sarcolemma destabilization and destruction. Lowering the PE content of myocytes, especially of the sarcolemma, is then expected to attenuate myocyte damage after simulated ischemia or metabolic inhibition. Culturing neonatal rat heart myocytes in the presence of N,N-dimethylethanolamine resulted in the synthesis of the bilayer-preferring N,N-dimethyl-PE and a lowering of the ratio between nonbilayer- and bilayer-preferring phospholipids from 0.58 to 0.30. This change in phospholipid composition did not impair cell functioning but did result in a strong attenuation of cell damage on ischemia or metabolic inhibition. A good correlation between the nonbilayer-preferring phospholipid content and the degree of cell damage was obtained (r = 0.98). These results provide further evidence that physicochemical properties of the sarcolemmal phospholipids play a crucial role in the sarcolemmal disruption during prolonged ischemia and/or reperfusion.
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