Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that similar to 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin ...
详细信息
Image correlation spectroscopy and cross correlation spectroscopy were used to demonstrate that similar to 25% of the internalization-competent influenza virus hemagglutinin mutant, HA+8, is colocalized with clathrin and AP-2 at the plasma membrane of intact cells, while wild-type HA (which is excluded from coated pits) does not colocalize with either protein. Clathrin and AP-2 clusters were saturated when HA+8 was overexpressed, and this was accompanied by a redistribution of AP-2 into existing coated pits. However, de novo coated pit formation was not observed. In nontreated cells, the number of clusters of clathrin or AP-2 colocalized with HA+8 was always comparable. Hypertonic treatment which disperses the clathrin lattices resulted in more clusters containing AP-2 and HA+8 than clathrin and HA+8, Less colocalization of HA+8 with clathrin was also observed after cytosol acidification, which causes the formation of deeply invaginated pits, where the HA+8 may be inaccessible to extracellular labeling by antibodies, and blocks coated vesicle budding. However, cytosol acidification elevated the number of clusters containing both HA+8 and AP-2, suggesting an increase in their level of association outside of the deep invaginations. Our results imply that AP-2 and HA+8 can colocalize in clusters devoid of clathrin, at least in cells treated to alter the clathrin lattice structure. Although we cannot ascertain whether this also occurs in untreated cells, we propose that AP-2 binding to membrane proteins carrying internalization signals can occur prior to the binding of AP-2 to clathrin. While such complexes can in principle serve to recruit clathrin for the formation of new coated pits, the higher affinity of the internalization signals for clathrin-associated AP-2 [Rapoport, I., et al, (1997) EMBO J, 16, 2240-2250] makes it more likely that once the AP-2-membrane protein complexes form, they are quickly recruited into existing coated pits.
Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic, However, the function of acidification in individual compartments has been dif...
详细信息
Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic, However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects, To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments, We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport, Al low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from the trans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin, At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes, Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases, Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell, The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport.
The homotetrameric M-2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M-2 transmembrane domain is believed to play an important role i...
详细信息
The homotetrameric M-2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M-2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine, A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals, Thus, membrane currents of oocytes of Xenopus laevis expressing the M-2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu2+ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (K-diss similar to 2 mu M) that lies deep within the pore and shows rather slow-binding kinetics (k(on) = 18.6 +/- 0.9 M-1 s(-1)). The pH dependence of the interaction with the high affinity Cu2+-binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site Lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu2+ could be prevented by prior application of the reversible blocker of M-2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M-2. Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu2+. Binding of Cu2+ the high affinity site resulted in an app
The NS1A protein of influenza A virus specifically inhibits the cellular machinery that processes the 3' ends of cellular pre-mRNAs by targeting two of the essential proteins of this machinery. Because the virus d...
详细信息
The NS1A protein of influenza A virus specifically inhibits the cellular machinery that processes the 3' ends of cellular pre-mRNAs by targeting two of the essential proteins of this machinery. Because the virus does not use this cellular machinery to synthesize the 3' poly(A) ends of viral mRNA, the nuclear export of cellular but not viral mRNAs is selectively inhibited.
The transfer of radioactive cholesterol from influenza virus to excess phosphatidylcholine-cholesterol vesicles was studied. Viral cholesterol was found in 2 pools, 1 rapidly exchangeable with vesicles, the other slow...
详细信息
The transfer of radioactive cholesterol from influenza virus to excess phosphatidylcholine-cholesterol vesicles was studied. Viral cholesterol was found in 2 pools, 1 rapidly exchangeable with vesicles, the other slowly or not at all exchangeable. The rapidly exchangeable pool probably corresponds to cholesterol present on the outer surface of the viral bilayer;the slowly exchangeable pool probably corresponds to inner surface cholesterol. Approximately equal amounts are present in each pool, suggesting that cholesterol distribution is not markedly asymmetric in the viral bilayer. A half-time for the rate of equilibration between the 2 sides of the bilayer (flip-flop) was .apprx. 13 days at ***. C with a 90% confidence interval of 3.4-.infin. days. Similar experiments were carried out which followed the time course of transfer of labeled phospholipids from the viral bilayer to phospholipid vesicles, catalyzed by phospholipid exchange protein from beef heart. The half-times for the flip-flop of phosphatidylcholine and spingomyelin were indeterminately in excess of 10 and 30 days at ***. C respectively. Exchange of the major components of the viral bilayer between the 2 surfaces apparently occurs very slowly relative to the turnover times of most membrane constituents. A plausible mechanism for the maintenance of membrane asymmetry [in the viral particle and in the host cell plasma membrane] over biologically relevant time periods is provided.
暂无评论