A novel function of karyopherin beta3 associated with apolipoprotein A-I secretion

Human karyopherin beta3, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin beta3 complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin beta3 and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin beta3 and apo A-I. In addition, overexpression of karyopherin beta3 significantly increased apo A-I secretion. These results suggest that karyopherin beta3 plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin beta3 and coronary artery diseases associated with apo A-I.     

Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption

Human plasma high-density lipoproteins (HDL) are important vehicles in reverse cholesterol transport, the cardioprotective mechanism by which peripheral tissue-cholesterol is transported to the liver for disposal. HDL is the target of serum opacity factor (SOF), a substance produced by Streptococcus pyogenes that turns mammalian serum cloudy. Using a recombinant (r) SOF, we studied opacification and its mechanism. rSOF catalyzes the partial disproportionation of HDL into a cholesteryl ester-rich microemulsion (CERM) and a new HDL-like particle, neo HDL, with the concomitant release of lipid-free (LF)-apo A-I. Opacification is unique; rSOF transfers apo E and nearly all neutral lipids of approximately 100,000 HDL particles into a single large CERM whose size increases with HDL-CE content (r approximately 100-250 nm) leaving a neo HDL that is enriched in PL (41%) and protein (48%), especially apo A-II. rSOF is potent; within 30 min at 37 degrees C, 10 nM rSOF opacifies 4 microM HDL. At respective low and high physiological HDL concentrations, LF-apo A-I is monomeric and tetrameric. CERM formation and apo A-I release have similar kinetics suggesting parallel or rapid sequential steps. According to the reaction products and kinetics, rSOF is a heterodivalent fusogenic protein that uses a docking site to displace apo A-I and bind to exposed CE surfaces on HDL; the resulting rSOF-HDL complex recruits additional HDL with its binding-delipidation site and through multiple fusion steps forms a CERM. rSOF may be a clinically useful and novel modality for improving reverse cholesterol transport. With apo E and a high CE content, CERM could transfer large amounts of cholesterol to the liver for disposal via the LDL receptor; neo HDL is likely a better acceptor of cellular cholesterol than HDL; LF-apo A-I could enhance efflux via the ATP-binding casette transporter ABCA1.     

Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [3H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma.     

Mechanism of dissociation of human apolipoprotein A-I from complexes with dimyristoylphosphatidylcholine as studied by guanidine hydrochloride denaturation

The reversibility of the binding of human apolipoprotein A-I (apo A-I) to phospholipid has been monitored through the influence of guanidine hydrochloride (Gdn-HCl) on the isothermal denaturation and renaturation of apo A-1/dimyristoylphosphatidylcholine (DMPC) complexes at 24 degree C. Denaturation was studied by incubating discoidal 1:100 and vesicular 1:500 mol/mol apo A-I/DMPC complexes with up to 7 M Gdn-HCl for up to 72 h. Unfolding of apo A-I molecules was observed from circular dichroism spectra while the distribution of protein between free and lipid-associated states was monitored by density gradient ultracentrifugation. The ability of apo A-I to combine with DMPC in the presence of Gdn-HCl at 24 degrees C was also investigated by similar procedures. In both the denaturation and renaturation of 1:100 and 1:500 complexes, the final values of the molar ellipticity and the ratio of free to bound apo A-I at various concentrations of Gdn-HCl are dependent on the initial state of the lipid and protein; apo A-I is more resistant to denaturation when Gdn-HCl is added to existing complexes than to a mixture of apo A-I and DMPC. There is an intermediate state in the denaturation pathway of apo A-I/DMPC complexes which is not present in the renaturation; the intermediate comprises partially unfold apo A-I molecules still associated with the complex by some of their apolar residues. Complete unfolding of the alpha helix and subsequent desorption of the apo A-I molecules from the lipid/water interface involve cooperative exposure of these apolar residues to the aqueous phase. The energy barrier associated with this desorption step makes the binding of apo A-I to DMPC a thermodynamically irreversible process. Consequently, binding constants of apo A-I and PC cannot be calculated simply from equilibrium thermodynamic treatments of the partitioning of protein between free and bound states. Apo A-I molecules do not exchange freely between the lipid-free and lipid-bound states, and extra work is required to drive protein molecules off the surface. The required increased in surface pressure can be achieved by a net mass transfer of protein to the surface; in vivo, increases in the surface pressure of lipoproteins by lipolysis can cause protein desorption.     

Speciation of human plasma high-density lipoprotein (HDL): HDL stability and apolipoprotein A-I partitioning

The distribution of apolipoprotein (apo) A-I between human high-density lipoproteins (HDL) and water is an important component of reverse cholesterol transport and the atheroprotective effects of HDL. Chaotropic perturbation (CP) with guanidinium chloride (Gdm-Cl) reveals HDL instability by inducing the unfolding and transfer of apo A-I but not apo A-II into the aqueous phase while forming larger apo A-I deficient HDL-like particles and small amounts of cholesteryl ester-rich microemulsions (CERMs). Our kinetic and hydrodynamic studies of the CP of HDL species separated according to size and density show that (1) CP mediated an increase in HDL size, which involves quasi-fusion of surface and core lipids, and release of lipid-free apo A-I (these processes correlate linearly), (2) >94% of the HDL lipids remain with an apo A-I deficient particle, (3) apo A-II remains associated with a very stable HDL-like particle even at high levels of Gdm-Cl, and (4) apo A-I unfolding and transfer from HDL to water vary among HDL subfractions with the larger and more buoyant species exhibiting greater stability. Our data indicate that apo A-I's on small HDL (HDL-S) are highly dynamic and, relative to apo A-I on the larger more mature HDL, partition more readily into the aqueous phase, where they initiate the formation of new HDL species. Our data suggest that the greater instability of HDL-S generates free apo A-I and an apo A-I deficient HDL-S that readily fuses with the more stable HDL-L. Thus, the presence of HDL-L drives the CP remodeling of HDL to an equilibrium with even larger HDL-L and more lipid-free apo A-I than with either HDL-L or HDL-S alone. Moreover, according to dilution studies of HDL in 3 M Gdm-Cl, CP of HDL fits a model of apo A-I partitioning between HDL phospholipids and water that is controlled by the principal of opposing forces. These findings suggest that the size and relative amount of HDL lipid determine the HDL stability and the fraction of apo A-I that partitions into the aqueous phase where it is destined for interaction with ABCA1 transporters, thereby initiating reverse cholesterol transport or, alternatively, renal clearance.     

Interaction of human apolipoprotein A-I with dipalmitoylphosphatidylcholine in vesicular and micellar complexes

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.     

Combined interaction of phospholipase C and apolipoprotein A-I with small unilamellar lecithin-cholesterol vesicles: influence of apolipoprotein A-I concentration and vesicle composition

We report the combined effects of phospholipase C (PLC), a pronucleating factor, and apolipoprotein A-I (apo A-I), an antinucleating factor, in solutions of model bile. Results indicate that apo A-I inhibits cholesterol nucleation from unilamellar lecithin vesicles by two mechanisms. Initially, inhibition is achieved by apo A-I shielding of hydrophobic diacylglycerol (DAG) moieties so as to prevent vesicle aggregation. Protection via shielding is temporary. It is lost when the DAG/apo A-I molar ratio exceeds a critical value. Subsequently, apo A-I forms small ( approximately 5-15 nm) complexes with lecithin and cholesterol that coexist with lipid-stabilized (400-800 nm) DAG oil droplets. This microstructural transition from vesicles to complexes avoids nucleation of cholesterol crystals and is a newly discovered mechanism by which apo A-I serves as an antinucleating agent in bile. The critical value at which a microstructural transition occurs depends on binding of apo A-I and so varies with the cholesterol mole fraction of vesicles. Aggregation of small, unilamellar, egg lecithin vesicles (SUVs) with varying cholesterol composition (0-60 mol %) was monitored for a range of apo A-I concentrations (2 to 89 microg/mL). Suppression of aggregation persists so long as the DAG-to-bound-apo A-I molar ratio is less than 100. A fluorescence assay involving dansylated lecithin shows that the suppression is an indirect effect of apo A-I rather than a direct inhibition of PLC enzyme activity. The DAG-to-total apo A-I molar ratio at which suppression is lost increases with cholesterol because of differences in apo A-I binding. Above this value, a microstructural transition to DAG droplets and lecithin/cholesterol A-I complexes occurs, as evidenced by sudden increases in turbidity and size and enhancement of Forster resonance energy transfer; structures are confirmed by cryo TEM.     

Expression and purification of recombinant apolipoprotein A-I Zaragoza (L144R) and formation of reconstituted HDL particles

Apolipoprotein A-I Zaragoza (L144R) (apo A-I Z), has been associated with severe hypoalphalipoproteinemia and an enhanced effect of high density lipoprotein (HDL) reverse cholesterol transport. In order to perform further studies with this protein we have optimized an expression and purification method of recombinant wild-type apo A-I and apo A-I Z and produced mimetic HDL particles with each protein. An pET-45 expression system was used to produce N-terminal His-tagged apo A-I, wild-type or mutant, in Escherichia coli BL21 (DE3) which was subsequently purified by affinity chromatography in non-denaturing conditions. HDL particles were generated via a modified sodium cholate method. Expression and purification of both proteins was verified by SDS-PAGE, MALDI-TOF MS and immunochemical procedures. Yield was 30mg of purified protein (94% purity) per liter of culture. The reconstituted HDL particles checked via non-denaturing PAGE showed high homogeneity in their size when reconstituted both with wild-type apo A-I and apo A-I Z. An optimized system for the expression and purification of wild-type apo A-I and apo A-I Z with high yield and purity grade has been achieved, in addition to their use in reconstituted HDL particles, as a basis for further studies.     

Apolipoproteins A-I,A-II and E are independently distributed among intracellular and newly secreted HDL of human hepatoma cells

Whereas hepatocytes secrete the major human plasma high density lipoproteins (HDL)-protein, apo A-I, as lipid-free and lipidated species, the biogenic itineraries of apo A-II and apo E are unknown. Human plasma and HepG2 cell-derived apo A-II and apo E occur as monomers, homodimers and heterodimers. Dimerization of apo A-II, which is more lipophilic than apo A-I, is catalyzed by lipid surfaces. Thus, we hypothesized that lipidation of intracellular and secreted apo A-II exceeds that of apo A-I, and once lipidated, apo A-II dimerizes. Fractionation of HepG2 cell lysate and media by size exclusion chromatography showed that intracellular apo A-II and apo E are fully lipidated and occur on nascent HDL and VLDL respectively, while only 45% of intracellular apo A-I is lipidated. Secreted apo A-II and apo E occur on small HDL and on LDL and large HDL respectively. HDL particles containing both apo A-II and apo A-I form only after secretion from both HepG2 and Huh7 hepatoma cells. Apo A-II dimerizes intracellularly while intracellular apo E is monomeric but after secretion associates with HDL and subsequently dimerizes. Thus, HDL apolipoproteins A-I, A-II and E have distinct intracellular and post-secretory pathways of hepatic lipidation and dimerization in the process of HDL formation. These early forms of HDL are expected to follow different apolipoprotein-specific pathways through plasma remodeling and reverse cholesterol transport.     

Effects of apolipoproteins on the kinetics of cholesterol exchange

The effects of apolipoproteins on the kinetics of cholesterol exchange have been investigated by monitoring the transfer of [14C]cholesterol from donor phospholipid/cholesterol complexes containing human apolipoproteins A, B, or C. Negatively charged discoidal and vesicular particles containing purified apolipoproteins complexed with lipid (75 mol % egg PC, 15 mol % dicetyl phosphate, and 10 mol % cholesterol) and a trace of [14C]cholesterol were incubated with a 10-fold excess of neural, acceptor, small unilamellar vesicles (SUV; 90 mol % egg PC and 10 mol % cholesterol). The donor and acceptor particles were separated by chromatography on DEAE-Sepharose, and the rate of movement of labeled cholesterol was analyzed as a first-order exchange process. The kinetics of exchange of cholesterol from both vesicular and discoidal complexes that contain apoproteins are consistent with an aqueous diffusion mechanism, as has been established previously for PC/cholesterol SUV. The addition of 2-3 molecules of apo A-I to a donor SUV does not significantly alter the half-time (t1/2), which is 80 +/- 9 min at 37 degrees C. However, addition of 5-12 apo A-I molecules progressively decreases t1/2 from 65 +/- 2 to 45 +/- 4 min. This enhancement in the rate of desorption of cholesterol molecules is presumed to arise from the creation of packing defects at boundaries around the apoprotein molecules, which are intercalated among the phospholipid and cholesterol molecules in the surface of the donor SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.     

Human apolipoprotein A-I forms small high density lipoprotein particles in rats in vivo.

The effects of injection of purified human or rat apolipoprotein (apo) A-I (1.7 mg/100 g body weight) on the size and composition of rat high density lipoprotein (HDL) particles have been investigated. The injection of human apo A-I results in the formation (over a period of 3 to 6 h) of a population of smaller HDL particles resembling human HDL3. This population of smaller particles contains human apo A-I and rat apo A-IV but lacks rat apo A-I and rat apo E. Small HDL3-like particles are not detected in rat plasma following the injection of rat apo A-I. Associated with the injection of either human or rat apo A-I is a gradual increase of plasma cholesterol levels of 20 to 50% (over 24 h) and the appearance of larger HDL particles. The results suggest that the smaller HDL particles in human plasma compared to rat plasma are not simply due to the action of lipid modifying enzymes or lipid transfer proteins but a specific property of human apo A-I.     

Rapid regulation of apolipoprotein A-I secretion in HepG2 cells by a factor associated with bovine high-density lipoproteins

The effect of fetal bovine serum (FBS) on the secretion of apolipoprotein A-I (apo A-I) by HepG2 cells was studied. The cells incubated with FBS always secreted more apo A-I than the cells incubated with serum-free medium. The changes in the rate of apo A-I secretion were observed within 1 h after addition or depletion of serum. The high-density lipoproteins (HDL) or the lipoprotein-deficient serum (LPDS) obtained from FBS also stimulated apo A-I secretion rapidly to the same level as obtained with FBS. Addition of low-density lipoproteins did not have any effect. The rate of general protein synthesis was not affected by short-term incubations with or without serum or HDL. The rate of apolipoprotein E secretion by these cells did not change significantly, parallel to the changes in apo A-I secretion in the presence or absence of FBS. It is concluded that serum may have a factor that plays a specific role in the regulation of apo A-I secretion by the liver cells and this factor is associated with the HDL fraction.     

The origin and metabolism of a nascent pre-β high density lipoprotein involved in cellular cholesterol efflux

The pre-β HDL fraction constitutes a heterogeneous population of discoid nascent HDL particles. They transport from 1 to 25 % of total human plasma apo A-I. Pre-β HDL particles are generated de novo by interaction between ABCA1 transporters and monomolecular lipid-free apo A-I. Most probably, the binding of apo A-I to ABCA1 initiates the generation of the phospholipid-apo A-I complex which induces free cholesterol efflux. The lipid-poor nascent pre-β HDL particle associates with more lipids through exposure to the ABCG1 transporter and apo M. The maturation of pre-β HDL into the spherical α-HDL containing apo A-I is mediated by LCAT, which esterifies free cholesterol and thereby forms a hydrophobic core of the lipoprotein particle. LCAT is also a key factor in promoting the formation of the HDL particle containing apo A-I and apo A-II by fusion of the spherical α-HDL containing apo A-I and the nascent discoid HDL containing apo A-II. The plasma remodelling of mature HDL particles by lipid transfer proteins and hepatic lipase causes the dissociation of lipid-free/lipid-poor apo A-I, which can either interact with ABCA1 transporters and be incorporated back into pre-existing HDL particles, or eventually be catabolized in the kidney. The formation of pre-β HDL and the cycling of apo A-I between the pre-β and α-HDL particles are thought to be crucial mechanisms of reverse cholesterol transport and the expression of ABCA1 in macrophages may play a main role in the protection against atherosclerosis.     

Apo A-II participates in HDL–liposome interaction by the formation of new pre-β mobility particles and the modification of liposomes

Interaction between high density lipoproteins (HDL) and liposomes results in both a structural modification of HDL and the generation of new pre-β HDL-like particles. Here, phosphatidylcholine liposomes and human HDL were incubated at liposomal phospholipid/HDL phospholipid (L-PL/HDL-PL) ratios of 1:1, 3:1 and 5:1 with a subsequent assessment of the distribution of apolipoprotein (apo) A-I, apo A-II, free cholesterol (FC) and PL between newly generated pre-β mobility lipoproteins and non-disrupted liposomes. Both at L-PL/HDL-PL ratios of 3:1 and 5:1 the fraction of liposomal-derived PL associated with pre-β fraction was significantly higher than those accepted by α-HDL. We found that 78% of apo A-I released from HDL was incorporated into pre-β mobility fraction. The relative contents of PL and apo A-I in pre-β fraction were constant irrespective of the initial L-PL/HDL-PL ratio in the incubation mixture and accounted for approximately 83 and 11%, respectively. Apo A-II was detached from HDL to a similar extent as apo A-I and distributed evenly between pre-β fraction and non-disrupted liposomes. Apo A-II constituted approximately 1%, by weight, in these fractions at all L-PL/HDL-PL ratios investigated. It corresponded approximately to 10% of pre-β fraction protein mass. Both liposomes and pre-β fraction accepted comparable amounts of FC released from HDL. This data indicated that during the interaction between human HDL and phosphatidylcholine liposome apo A-II participates both in structural modification of liposomes and in the generation of pre-β mobility fraction of constant content of PL, apo A-I and apo A-II. Involvement of apo A-II in HDL–liposome interaction may influence the anti-atherogenic properties of liposomes.     

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

How peptide hormone vesicles are transported to the secretion site for exocytosis

Post-Golgi transport of peptide hormone-containing vesicles from the site of genesis at the trans-Golgi network to the release site at the plasma membrane is essential for activity-dependent hormone secretion to mediate various endocrinological functions. It is known that these vesicles are transported on microtubules to the proximity of the release site, and they are then loaded onto an actin/myosin system for distal transport through the actin cortex to just below the plasma membrane. The vesicles are then tethered to the plasma membrane, and a subpopulation of them are docked and primed to become the readily releasable pool. Cytoplasmic tails of vesicular transmembrane proteins, as well as many cytosolic proteins including adaptor proteins, motor proteins, and guanosine triphosphatases, are involved in vesicle budding, the anchoring of the vesicles, and the facilitation of movement along the transport systems. In addition, a set of cytosolic proteins is also necessary for tethering/docking of the vesicles to the plasma membrane. Many of these proteins have been identified from different types of (neuro)endocrine cells. Here, we summarize the proteins known to be involved in the mechanisms of sorting various cargo proteins into regulated secretory pathway hormone-containing vesicles, movement of these vesicles along microtubules and actin filaments, and their eventual tethering/docking to the plasma membrane for hormone secretion.