Clathrin assembly protein AP180: primary structure, domain organization and identification of a clathrin binding site

Binding of AP180 to clathrin triskelia induces their assembly into 60-70 nm coats. The largest rat brain cDNA clone isolated predicts a molecular weight of 91,430 for AP180. Two cDNA clones have an additional small 57 bp insert. The deduced molecular weight agrees with gel filtration results provided the more chaotropic denaturant 6 M guanidinium thiocyanate is substituted for the weaker guanidinium chloride. The sequence and the proteolytic cleavage pattern suggest a three domain structure. The N-terminal 300 residues (pI 8.7) harbour a clathrin binding site. An acidic middle domain (pI 3.6, 450 residues), interrupted by an uncharged alanine rich segment of 59 residues, appears to be responsible for the anomalous physical properties of AP180. The C-terminal domain (166 residues) has a pI of 10.4. AP180 mRNA is restricted to neuronal sources. AP180 shows no significant homology to known clathrin binding proteins, but is nearly identical to a mouse phosphoprotein (F1-20). This protein, localized to synaptic termini, has so far been of unknown function.     

Polyhedral assembly of a membrane protein in its three-dimensional crystal

A novel ordered assemblage of bacteriorhodopsin, a transmembrane protein functioning as a light-driven proton pump, is found in its three-dimensional crystal. Atomic force microscope images of the crystal surface reveal that spherical protein clusters with a diameter of approximately 50 nm are hexagonally close-packed. Electron micrographs of mechanically disintegrated crystals show that the inside of the protein cluster is filled with the mother liquor. The crystal is made up of hollow protein clusters. When disintegrated crystals are illuminated in the presence of a lipophilic anion, a significant alkalization of the external medium occurs. This result indicates that the protein cluster contains native lipids and that the cytoplasmic side of the protein faces the external medium. X-ray diffraction patterns and the observed diameter of the spherical shell suggest that approximately 200 bacteriorhodopsin trimers are aligned on a polyhedral surface lattice. Another remarkable feature of the spherical assemblies of bacteriorhodopsin is that they fuse with each other at low ionic strength and occasionally form a tubular or doughnut-like structure. The concept of membrane protein polymorphism is introduced on the basis of these observations, and it is used to describe the dynamic structure of some other biological membranes.     

Testing the charge difference hypothesis for the assembly of a eucaryotic multispanning membrane protein

The Glut1 glucose transporter is a glycoprotein whose membrane topology has been verified by a number of experimental observations, all of which are consistent with a 12-transmembrane helix model originally based on hydrophobicity analysis. We used Glut1 as a model multispanning membrane protein to test the Charge Difference Hypothesis (Hartmann, E., Rapoport, T. A., and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5786-5790), which asserts that the topology of a eucaryotic multispanning membrane protein is determined solely by the amino acid charge difference across the first transmembrane segment. The charge difference across the first transmembrane segment of Glut1 was progressively inverted in two independent series of mutants, one series in which only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number of negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first transmembrane segment does affect the topology of the protein, but that contrary to the hypothesis, it only dictates the orientation of the first transmembrane segment and the disposition of the amino terminus and the first linker domain. Charge inversion resulted in the formation of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane. The topology of downstream regions of Glut1 was unaffected by charge inversion across the first transmembrane segment, indicating that downstream sequences are important in determining the local topological disposition of the molecule.     

Lipid patches in membrane protein oligomers: crystal structure of the bacteriorhodopsin-lipid complex

Heterogenous nucleation on small molecule crystals causes a monoclinic crystal form of bacteriorhodopsin (BR) in which trimers of this membrane protein pack differently than in native purple membranes. Analysis of single crystals by nano-electrospray ionization-mass spectrometry demonstrated a preservation of the purple membrane lipid composition in these BR crystals. The 2.9-A x-ray structure shows a lipid-mediated stabilization of BR trimers where the glycolipid S-TGA-1 binds into the central compartment of BR trimers. The BR trimer/lipid complex provides an example of local membrane thinning as the lipid head-group boundary of the central lipid patch is shifted by 5 A toward the membrane center. Nonbiased electron density maps reveal structural differences to previously reported BR structures, especially for the cytosolic EF loop and the proton exit pathway. The terminal proton release complex now comprises an E194-E204 dyad as a diffuse proton buffer.     

Membrane assembly of the Escherichia coli outer membrane protein OmpA: exploring sequence constraints on transmembrane beta-strands

The eight-stranded antiparallel beta-barrel domain of the OmpA protein from Escherichia coli serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. Previous studies have shown that neither the periplasmic turns nor the surface-exposed loops contain topogenic information. Consequently, the question of whether any structural constraint is imposed onto individual transmembrane beta-strands is now addressed. To this end, amino acid sequences of beta-strands 4, 6 and 8 were randomized. In vivo membrane assembly of mutant proteins was assayed and 288 variants were sequenced. Three parameters were found to be important for efficient membrane assembly. (i) At least four of five randomized residues with side-chains pointing towards the lipid bilayer must be hydrophobic and none of the three central residues must be charged. (ii) Side-chains pointing into the beta-barrel interior must not be enlarged too much, possibly because of packing constraints. (iii) Proline residues are, in general, hardly tolerated in the transmembrane beta-strands.     

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.     

Studies of asymmetric membrane assembly

The major capsid protein of M13 bacteriophage is incorporated at each stage of infection into the host plasma membrane with its amino terminus exposed on the outer surface. Purified M13 coat protein is incorporated with the same asymmetry into synthetic phosphatidylcholine vesicles formed near the Tm of the lipid by a cholate dilution technique. We now report that the lipid in the pre-dilution mixture exists as mixed micelles of uniform size. Prior to dilution, the coat protein is present in at least two states of aggregation, both of which behave similarly in the model membrane assembly reaction. No detectable lipid-protein interaction occurs prior to dilution. Upon dilution there is rapid production of small closed vesicles and coat protein is converted to a chymotrypsin-resistant form, presumably reflecting its incorporation into these vesicle bilayers. Formation of large (greater than 6000 A diameter) vesicles occurs slowly with preservation of coat protein asymmetry and internal volume. A model for this assembly reaction is proposed.     

The structure of the membrane protein squalene-hopene cyclase at 2.0 A resolution

Squalene cyclases catalyze a cationic cyclization cascade, which is homologous to a key step in cholesterol biosynthesis. The structure of the enzyme from Alicyclobacillus acidocaldarius has been determined in a new crystal form at 2.0 A resolution (1 A=0.1 nm) and refined to an R-factor of 15.3 % (Rfree=18.7 %). The structure indicates how the initial protonation and the final deprotonation of squalene occur and how the transient carbocations are stabilized. The pathways of the flexible educt squalene from the membrane interior to the active center cavity and of the rigid fused-ring product hopene in the reverse direction are discussed. The enzyme contains eight so-called QW-sequence repeats that fortify the alpha/alpha-barrels by an intricate interaction network. They are unique to the known triterpene cyclases and are presumed to shield these enzymes against the released enthalpy of the highly exergonic catalyzed reaction. The enzyme is a monotopic membrane protein, the membrane-binding interactions of which are described and compared with those of two prostaglandin-H2 synthase isoenzymes, the only other structurally characterized proteins of this type. In the crystals the membrane-binding regions face each other, suggesting a micelle-type detergent structure between them.     

Apoprotein structure in the LH2 complex from Rhodopseudomonas acidophila strain 10050: modular assembly and protein pigment interactions

The refined structure of the peripheral light-harvesting complex from Rhodopseudomonas acidophila strain 10050 reveals a membrane protein with protein-protein interactions in the trans-membrane region exclusively of a van der Waals nature. The dominant factors in the formation of the complex appear to be extramembranous hydrogen bonds (suggesting that each apoprotein must achieve a fold close to its final structure in order to oligomerize), protein-pigment and pigment-pigment interactions within the membrane-spanning region. The pigment molecules are known to play an important role in the formation of bacterial light-harvesters, and their extensive mediation of structural contacts within the membrane bears this out. Amino acid residues determining the secondary structure of the apoproteins influence the oligomeric state of the complex. The assembly of the pigment array is governed by the apoproteins of LH2. The particular environment of each of the pigment molecules is, however, influenced directly by few protein contacts. These contacts produce functional effects that are not attributable to a single cause, e.g. the arrangement of an overlapping cycle of chromophores not only provides energy delocalisation and storage properties, but also has consequences for oligomer size, pigment distortion modes and pigment chemical environment, all of which modify the precise function of the complex. The evaluation of site energies for the pigment array requires the consideration of a number of effects, including heterogeneous pigment distortions, charge distributions in the local environment and mechanical interactions.     

Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal protein filensin represents a novel class of intermediate filament protein

The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.     

The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.     

Bacterial protein kinases that recognize tertiary rather than primary structure?

While all characterized eukaryotic protein kinases that phosphorylate hydroxy aminoacyl residues in proteins recognize primary structure, certain bacterial protein kinases are proving to recognize tertiary structure. It is proposed that these latter enzymes evolved independently of the superfamily of the former protein kinases and that their modes of target protein recognition and action are entirely different.     

Toward understanding the assembly and structure of KATP channels

Adenosine 5'-triphosphate-sensitive potassium (KATP) channels couple metabolic events to membrane electrical activity in a variety of cell types. The cloning and reconstitution of the subunits of these channels demonstrate they are heteromultimers of inwardly rectifying potassium channel subunits (KIR6.x) and sulfonylurea receptors (SUR), members of the ATP-binding cassette (ABC) superfamily. Recent studies indicate that SUR and KIR6.x associate with 1:1 stoichiometry to assemble a large tetrameric channel, (SUR/KIR6.x)4. The KIR6.x subunits form the channel pore, whereas SUR is required for activation and regulation. Two KIR6.x genes and two SUR genes have been identified, and combinations of subunits give rise to KATP channel subtypes found in pancreatic beta-cells, neurons, and cardiac, skeletal, and smooth muscle. Mutations in both the SUR1 and KIR6.2 genes have been shown to cause familial hyperinsulinism, indicating the importance of the pancreatic beta-cell channel in the regulation of insulin secretion. The availability of cloned KATP channel genes opens the way for characterization of this family of ion channels and identification of additional genetic defects.     

Effects of exercise on dietary protein requirements

This paper reviews the factors (exercise intensity, carbohydrate availability, exercise type, energy balance, gender, exercise training, age, and timing of nutrient intake or subsequent exercise sessions) thought to influence protein need. Although there remains some debate, recent evidence suggests that dietary protein need increases with rigorous physical exercise. Those involved in strength training might need to consume as much as 1.6 to 1.7 g protein x kg(-1) x day(-1) (approximately twice the current RDA) while those undergoing endurance training might need about 1.2 to 1.6 g x kg(-1) x day(-1) (approximately 1.5 times the current RDA). Future longitudinal studies are needed to confirm these recommendations and asses whether these protein intakes can enhance exercise performance. Despite the frequently expressed concern about adverse effects of high protein intake, there is no evidence that protein intakes in the range suggested will have adverse effects in healthy individuals.     

Structure of the Drosophila projectin protein: isoforms and implication for projectin filament assembly

The rare alleles model of mutation-selection balance (MSB) hypothesis for the maintenance of genetic variation was evaluated for two quantitative traits, ovariole number and body size. Mutational variances (VM) for these traits, estimated from mutation accumulation lines, were 4.75 and 1.97 x 10(-4) times the environmental variance (VE), respectively. The mutation accumulation lines were studied in three environments to test for genotype x environment interaction (GEI) of new mutations; significant mutational GEI was found for both traits. Mutations for ovariole number have a quadratic relationship with competitive fitness, suggesting stabilizing selection for the trait; there is no significant correlation between mutations for body size and competitive fitness. Under MSB, the ratio of segregating genetic variance, VG, to mutational variance, VM, estimates the inverse of the selection coefficient against a heterozygote for a new mutation. Estimates of VG/VM for ovariole number and body size were both approximately 1.1 x 10(4). Thus, MSB can explain the level of variation, if mutations affecting these traits are under very weak selection, which is inconsistent with the empirical observation of stabilizing selection, or if the estimate of VM is biased downward by two orders of magnitude. GEI is a possible alternative explanation.     

Preprotein translocase of the outer mitochondrial membrane: molecular dissection and assembly of the general import pore complex

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of approximately 400,000 ( approximately 400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of approximately 100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.