The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding

Using a murine challenge model, we previously determined that human papillomavirus (HPV) pseudovirions initially bind preferentially to the cervicovaginal basement membrane (BM) at sites of trauma. We now report that the capsids undergo a conformational change while bound to the BM that results in L2 cleavage by a proprotein convertase (PC), furin, and/or PC5/6, followed by the exposure of an N-terminal cross-neutralization L2 epitope and transfer of the capsids to the epithelial cell surface. Prevention of this exposure by PC inhibition results in detachment of the pseudovirions from the BM and their eventual loss from the tissue, thereby preventing infection. Pseudovirions whose L2 had been precleaved by furin can bypass the PC inhibition of binding and infectivity. Cleavage of heparan sulfate proteoglycans (HSPG) with heparinase III prevented infection and BM binding by the precleaved pseudovirions, but did not prevent them from binding robustly to cell surfaces. These results indicate that the infectious process has evolved so that the initial steps take place on the BM, in contrast to the typical viral infection that is initiated by binding to the cell surface. The data are consistent with a dynamic model of in vivo HPV infection in which a conformational change and PC cleavage on the BM allows transfer of virions from HSPG attachment factors to an L1-specific receptor on basal keratinocytes migrating into the site of trauma.     

Localization of different photosystems in separate regions of chloroplast membranes

The stoichiometric amounts and the photoactivity kinetics of photosystem I (PSI) and of the α and β components of photosystem II (PSII_α and PSII_β) were compared in spinach chloroplast membrane (thylakoid) fractions derived from appressed and nonappressed regions. Stroma-exposed thylakoid fractions from the nonappressed regions were isolated by differential centrifugation following a mechanical press treatment of the chloroplasts. Thylakoid vesicles derived mainly from the appressed membranes of grana were isolated by the aqueous polymer two-phase partition method. Stroma-exposed thylakoids were found to have a chlorophyll a/chlorophyll b ratio of 6.0 and a PSII_β/PSI reaction center ratio of 0.3. Kinetic analysis of system II photoactivity revealed the absence of PSII_α from stroma-exposed thylakoids. The photoactivity of system I in stroma-exposed thylakoids showed a single kinetic component identical to that of unfractionated chloroplasts, suggesting that PSI does not receive excitation energy from the PSII-chlorophyll ab light-harvesting complex. Thus, stroma-exposed thylakoids are significantly enriched in both PSI and PSII_β. Inside-out vesicles from the appressed membranes of grana-partition regions had a chlorophyll a/chlorophyll b ratio of 2.0 and a PSII/PSI reaction center ratio of 10.0. The photoactivity of system II showed the membranes of the grana-partition regions to be significantly enriched in PSII_α. We conclude that PSII_α is exclusively located in the membranes of the grana partitions while PSII_β and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles derived from grana partitions and the kinetic homogeneity of the PSI complex suggest total exclusion of P700 as a functional component in the membrane of the grana-partition region.     

An unusual signal peptide facilitates late steps in the biogenesis of a bacterial autotransporter

Bacterial autotransporters are proteins that use a C-terminal porin-like domain to facilitate the transport of an upstream "passenger domain" across the outer membrane. Although autotransporters are translocated across the inner membrane (IM) via the Sec pathway, some of them contain exceptionally long signal peptides distinguished by a unique N-terminal sequence motif. In this study, we used the Escherichia coli O157:H7 autotransporter EspP as a model protein to investigate the function of the unusual signal peptides. We found that removal of the N-terminal motif or replacement of the EspP signal peptide did not affect translocation of the protein across the IM. Remarkably, modification of the signal peptide caused EspP to misfold in the periplasm and blocked transport of the passenger domain across the outer membrane. Further analysis suggested that the EspP signal peptide transits slowly through the Sec machinery. Based on these results, we propose that the unusual signal peptides not only function as targeting signals, but also prevent misfolding of the passenger domain in the periplasm by transiently tethering it to the IM.     

Feedback activation of factor XI by thrombin does not occur in plasma

In this study, we tested the hypothesis that factor XI (FXI) activation occurs in plasma following activation of the extrinsic pathway by thrombin-mediated feedback activation. We used two different assays: (i) a direct measurement of activated FXI by ELISA and (ii) a functional assay that follows the activation of the coagulation cascade in the presence or absence of a FXI inhibiting antibody by monitoring thrombin activity. We failed to detect any FXI activation or functional contribution to the activation of the coagulation cascade in platelet poor or platelet-rich plasma, when activation was initiated by thrombin or tissue factor. Additionally, we found that, in the absence of a contact system inhibitor during blood draw, contact activation of FXI can mistakenly appear as thrombin- or tissue-factor-dependent activation. Thus, activation of FXI by thrombin in solution or on the surface of activated platelets does not appear to play a significant role in a plasma environment. These results call for reevaluation of the physiological role of the contact activation system in blood coagulation.     

Immunocytochemical localization of acyl-lipid desaturases in cyanobacterial cells: evidence that both thylakoid membranes and cytoplasmic membranes are sites of lipid desaturation

There are four acyl-lipid desaturases in the cyanobacterium Synechocystis sp. PCC 6803. Each of these desaturases introduces a double bond at a specific position, such as the Delta6, Delta9, Delta12, or omicron3 position, in C18 fatty acids. The localization of the desaturases in cyanobacterial cells was examined immunocytochemically with antibodies raised against synthetic oligopeptides that corresponded to the carboxyl-terminal regions of the desaturases. All four desaturases appeared to be located in the regions of both the cytoplasmic and the thylakoid membranes. These findings suggest that fatty acid desaturation of membrane lipids takes place in the thylakoid membranes as well as in the cytoplasmic membranes.     

Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) delta to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPARdelta.     

Calmodulin-binding proteins in plasma membranes from adrenocortical cells

Highly purified plasma membranes from Y-1 mouse adrenal tumor cells and those from bovine fasciculata cells were shown by [125I]iodocalmodulin overlay to contain five calmodulin-binding proteins of 240,000, 150,000, 66,000, 60,000, and 51,000 mol wt (Mr). Three of these proteins were also detected by affinity chromatography on calmodulin-Sepharose. Calmodulin binding was inhibited by competition with unlabeled calmodulin and by an inhibitor of calmodulin (trifluoperazine). Binding to each of the proteins was Ca2+ dependent. The relative proportion of binding to each of the five proteins was very different for Y-1 and bovine membranes. In Y-1 membranes as much as 50% of total binding was to the 51,000 Mr protein, whereas in bovine membranes more than 50% of binding occurred with the 150,000 Mr protein. Three of the five proteins were tentatively identified as follows: the 240,000 Mr protein is alpha-spectrin, the 60,000 Mr protein is the A subunit of the Ca2+/calmodulin-dependent protein phosphatase called calcineurin and the 51,000 Mr protein is the major subunit of a Ca2+/calmodulin-dependent protein kinase. The kinase was shown to act on specific substrates. It is concluded that calmodulin, by binding to the kinase and phosphatase, is capable of influencing the degree of phosphorylation of specific substrates in the plasma membranes of adrenal cells, and by binding to alpha-spectrin it may influence the cytoskeletons of these cells. These effects of calmodulin are likely to be important in the regulation of steroid synthesis in the adrenal cortex.     

Order of lipid phases in model and plasma membranes

Lipid rafts are nanoscopic assemblies of sphingolipids, cholesterol, and specific membrane proteins that contribute to lateral heterogeneity in eukaryotic membranes. Separation of artificial membranes into liquid-ordered (Lo) and liquid-disordered phases is regarded as a common model for this compartmentalization. However, tight lipid packing in Lo phases seems to conflict with efficient partitioning of raft-associated transmembrane (TM) proteins. To assess membrane order as a component of raft organization, we performed fluorescence spectroscopy and microscopy with the membrane probes Laurdan and C-laurdan. First, we assessed lipid packing in model membranes of various compositions and found cholesterol and acyl chain dependence of membrane order. Then we probed cell membranes by using two novel systems that exhibit inducible phase separation: giant plasma membrane vesicles [Baumgart et al. (2007) Proc Natl Acad Sci USA 104:3165–3170] and plasma membrane spheres. Notably, only the latter support selective inclusion of raft TM proteins with the ganglioside GM1 into one phase. We measured comparable small differences in order between the separated phases of both biomembranes. Lateral packing in the ordered phase of giant plasma membrane vesicles resembled the Lo domain of model membranes, whereas the GM1 phase in plasma membrane spheres exhibited considerably lower order, consistent with different partitioning of lipid and TM protein markers. Thus, lipid-mediated coalescence of the GM1 raft domain seems to be distinct from the formation of a Lo phase, suggesting additional interactions between proteins and lipids to be effective.     

Preliminary characterization of calcium binding in islet-cell plasma membranes

Summary Preliminary characterization of calcium binding was determined in a highly-enriched islet-cell plasma membrane fraction using a membrane filtration technique. Equilibrium calcium binding was specific, concentration dependent and saturable. Scatchard analysis indicated the existence of more than one class of calcium binding sites. The affinity constants and maximum binding capacities were 1.14 ×10⁵ M^-1 and 1.2 picomol/g protein and 1.17× 10³ M^-1 and 64.8 picomol/g for the high and low affinity sites, respectively. Bound ⁴⁵Ca²⁺ was dissociated from the plasma membranes in a biphasic manner in the presence of excess unlabelled calcium.     

Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers

For the act of membrane fusion, there are two competing, mutually exclusive molecular models that differ in the structure of the initial pore, the pathway for ionic continuity between formerly separated volumes. Because biological “fusion pores” can be as small as ionic channels or gap junctions, one model posits a proteinaceous initial fusion pore. Because biological fusion pore conductance varies widely, another model proposes a lipidic initial pore. We have found pore opening and flickering during the fusion of protein-free phospholipid vesicles with planar phospholipid bilayers. Fusion pore formation appears to follow the coalescence of contacting monolayers to create a zone of hemifusion where continuity between the two adherent membranes is lipidic, but not aqueous. Hypotonic stress, causing tension in the vesicle membrane, promotes complete fusion. Pores closed soon after opening (flickering), and the distribution of fusion pore conductance appears similar to the distribution of initial fusion pores in biological fusion. Because small flickering pores can form in the absence of protein, the existence of small pores in biological fusion cannot be an argument in support of models based on proteinaceous pores. Rather, these results support the model of a lipidic fusion pore developing within a hemifused contact site.     

The thyrotrophin receptor in human thyroid plasma membranes: effect of serum immunoglobulins.

A homologous receptor assay system using human thyroid membranes and 125I-labelled human TSH (hTSH) was used to study the effect of serum and serum fractions on the binding of [125I]hTSH to the membranes. Scatchard analysis showed a single population of binding sites for TSH. Gamma globulin fractions prepared from sera of patients with Graves' disease were able to displace [125I]hTSH from the membrane to a greater extent than normal gamma globulin in 21 out of 45 cases. Increased displacement activity was seen in patients with thyroiditis, hot nodules and euthyroid eye disease but not in patients with toxic multinodular goitres. Further fractionation of the gamma globulin fraction showed that the stimulatory activity was not confined to the IgG fraction. Scatchard plots showed gamma globulin fractions decreased the number of receptor sites available for TSH binding but did not alter the affinity of the receptor for TSH. IgG fractions showed different slopes and intercepts and appeared to decrease the affinity of the receptor for TSH. LATS activity in human serum may be explained on the basis of these observations on the properties of the TSH receptor.     

The role of calmodulin in the responses to adrenocorticotropin of plasma membranes from adrenal cells

Inhibitors of calmodulin [trifluoperazine, chlorpromazine, pimozide, and calmidazolium N-(6-aminohexyl)5-chloro-1-napthalenesulphonamide (W7)] and calmodulin antibodies were used to investigate the role of calmodulin in the response of Y-1 mouse adrenal cells to ACTH, with particular reference to events in the plasma membrane. In whole cells it was found that two responses (production of steroids and cAMP) to two stimulating agents (ACTH and forskolin) were inhibited by trifluoperazine at concentrations consistent with those involved in binding of the inhibitor to pure calmodulin (10-25 microM). The steroidogenic responses were also inhibited by the three other inhibitors of calmodulin (chlorpromazine, calmidazolium, and W-7). Trifluoperazine and pimozide (1-500 microM) did not inhibit binding of an [125I]ACTH analog to highly purified plasma membranes of Y-1 cells or to the cells themselves. With Y-1 plasma membranes it was found that trifluoperazine, pimozide, W-7, and calmodulin antibodies inhibited the increase in adenylate cyclase activity in response to ACTH, but not the cyclase responses to cholera toxin or forskolin. Moreover, the effect of cholera toxin on the ADP-ribosylation of specific membrane substrates was independent of the presence or absence of endogenous and/or exogenous Ca2+/calmodulin. The response of adenylate cyclase to ACTH was also decreased in plasma membranes from which calmodulin was removed by washing, and exogenous calmodulin partly reversed this decrease. Anti-calmodulin immunoglobulin inhibited the stimulation of adenylate cyclase produced in plasma membrane by ACTH, but was without effect on the responses to cholera toxin and forskolin. Exogenous calmodulin partly reversed the inhibition of stimulation by ACTH of adenylate cyclase produced by the antibody. It is concluded that calmodulin influences the events taking place in the plasma membrane in response to ACTH, after the binding of the hormone to its receptor and before the action of the G protein (Gs). That is, calmodulin is involved in coupling the occupied receptor to Gs. The effects of inhibitors of calmodulin in whole cells must involve some additional effect(s) requiring the intact cell.