Protein translocation across biological membranes

Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work. Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work.     

Protein conformations in the plasma membrane

Infrared spectroscopy and optical rotatory dispersion have been used to test theories of structure of membrane protein. No evidence has been found to support the view that adjacent to the lipid there is a monolayer of protein in the beta-conformation. The extracted protein appears to be a fairly typical globular protein with a low a-helical content.     

Structure of biological membranes

The combined x-ray diffraction and electron microscopic examination of myelin has provided reasonable, but not conclusive, support for its structure as a basically bimolecular leaflet of phospholipid that is partially interspersed with protein. But there is very little basis for extending this concept to biological membranes in general. There is no adequate experimental support for the specific orientation of phospholipids as proposed in the unit membrane theory or for the proposed polar nature of protein-lipid bonds, even in myelin. Membranes differ widely in chemical composition, metabolism, function, enzymatic composition, and even in their electron microscopic image. The only similarity is their general resemblance in electron micrographs, but, until more is known about the chemistry of electron microscopy, this evidence cannot be interpreted with confidence. One positive conclusion to which I have come is that much more chemical evidence must, and can, be obtained. Techniques for the isolation of membranes are improving and protein and lipid chemistry are now highly refined arts. Quantitative analysis of many different membranes is possible and the data can be related in some instances, notably bacterial plasma membranes, to calculations of surface area. Chemical and physical changes induced in membranes of widely different lipid composition by the preparatory procedures of electron microscopy can be determined directly and correlated with the electron microscopic image. Model systems can be assembled whose compositions closely resemble those of biological membranes. Membranes can be disassociated into subunits whose properties can be studied. In particular, x-ray diffraction analysis and electron microscopy by negative staining of reaggregates of lipoproteins isolated from membranes would be very informative. Perhaps most important, the problem of membrane structure must be considered in relation to the problems of membrane function and membrane biosynthesis.     

Protein translocation across membranes

Many newly synthesized proteins must be translocated across a membrane to reach their final destinations. Translocation requires a signal on the protein itself, a loose conformation of the protein, energy, and receptor-like components in the cytosol and on the target membrane.     

Electron diffraction of wet biological membranes

Electron diffraction patterns were obtained for the first time from single wet phospholipid bilayers and from wet human erythrocyte membranes by using a temperature-controlled electron microscope hydration stage. Selective area diffraction showed the existence of semicrystalline domains. A structural transition was observed at the transition temperature of the wet dipalmitoyl lecithin bilayer.     

Chloral hydrate: a solvent for biological membranes

A buffer system conitainin chloral hydrate, taurine, and bromopyridinium lactate was used to dissolve several biological membranes and separate their protein components by polyacrylamide gel electrophoresis. This solvent system was capable of separating molecules of similar size on the hasis of their charge and allows easy recovery of the proteins Thus. aqueous chloral hydrate is an effective solvent for biological membranes.     

Thermodynamic treatment of radio-tracer movements across biological membranes

A Beltsville Small White turkey poult of parthenogenetic origin hatched in the spring of 1958, matured, and produced semen containing viable spermatozoa. Semen from this male was used in January 1959 to inseminate seven virgin and seven previously mated Beltsville Small White turkey hens. Three hundred and twenty eggs were incubated, of which 175 or 54.7 percent were infertile. One hundred and twenty-two poults, about equally divided as to sex, hatched unaided from 145 fertile eggs.     

X-ray standing waves: a molecular yardstick for biological membranes

Structural information on an atomic scale has been obtained for a Langmuir-Blodgett (LB) trilayer system by means of long-period x-ray standing waves. The LB trilayer of zinc and cadmium arachidate was deposited on a layered synthetic microstructure (LSM) consisting of 200 tungsten/silicon layer pairs with a 25 A period. A 30 A thermally induced inward collapse of the zinc atom layer that was initially located in the LB trilayer at 53 A above the LSM surface has been observed. The mean position and width of the zinc atom layer was determined with a precision of +/- 0.3 A.     

Hyaluronates: relation between molecular conformations

The discovery that both potassium and sodium salts of hyaluronic acid can exist in a double-strand helical conformation that will convert to the already known single-strand helical structures illustrates the remarkable conformational versatility of this biopolymer. X-ray diffraction was used to monitor variations in molecular conformation as a function of several independent, controllable variables, such as relative humidity, temperature, and applied tension. A scheme is presented for the interrelation of a range of hyaluronate conformations.     

Raman spectroscopic investigation of gramicidin A' conformations

Gramicidin A' is believed to form transmembrane channels in lipid bilayers and biological membranes. The first Raman spectroscopic study of gramicidin A' is presented. Evidence is found for two types of conformation. One type is found in the powder and has a Raman spectrum similar to that of model polypeptides with beta hydrogen bonding. The second type is found when gramicidin A' is dissolved in dimethyl sulfoxide.     

Ferroin-collodion membranes: dynamic concentration patterns in planar membranes

Patterns and waves of the Belousov-Zhabotinskii reaction are produced in membranes in which one reactant is immobilized. Convection is eliminated, the generation and deformation of wave forms are studied, and patterns are permnanently fixed. Wave shape, frequency, length, and phase velocity are explained theoretically by the interactions of diffusion with reaction.     

Doubls-helical polynucleotides: immunochemical recognition of differing conformations

Rabbit antibodies to double-helical RNA react by complement fixation with synthetic or natural double-strand RNA but not with native DNA. In turn, human (from systemic lupus erythematosus patients) antibodies to native DNA do not react with double-strand RNA. Both types of antibodies show cross-reactions (from 1 percent to 50 percent) with RNA-DNA hybrids, but antibodies to the hybrids do not react at all with double-strand RNA or with native DNA. Antibodies to polydeoxyguanylate.polydeoxycytidylate also failed to react with native DNA.     

Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase

In thioredoxin reductase (TrxR) from Escherichia coli, cycles of reduction and reoxidation of the flavin adenine dinucleotide (FAD) cofactor depend on rate-limiting rearrangements of the FAD and NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) domains. We describe the structure of the flavin-reducing conformation of E. coli TrxR at a resolution of 3.0 angstroms. The orientation of the two domains permits reduction of FAD by NADPH and oxidation of the enzyme dithiol by the protein substrate, thioredoxin. The alternate conformation, described by Kuriyan and co-workers, permits internal transfer of reducing equivalents from reduced FAD to the active-site disulfide. Comparison of these structures demonstrates that switching between the two conformations involves a "ball-and-socket" motion in which the pyridine nucleotide-binding domain rotates by 67 degrees.     

分子生物学在媒介生物研究中的应用

随着分子生物学及其技术迅猛发展,分子生物学已成为现代生物学研究领域的热点之一,在媒介生物研究中也得到了越来越多的应用,为媒介生物的分类鉴定提供了可测量的指标,对媒介生物的分类地位和进化关系有了更准确的定位.就分子生物学在媒介生物研究中应用的一些新进展做了综述,以期为媒介生物资源的检测开发利用、虫媒病毒的检测分析及所传疾病的诊断和防制策略提供理论依据.