The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.     

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F- actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.     

Statistical mechanics of protein folding by exhaustive enumeration

It is hard to construct theories for the folding of globular proteins because they are large and complicated molecules having enormous numbers of nonnative conformations and having native states that are complicated to describe. Statistical mechanical theories of protein folding are constructed around major simplifying assumptions about the energy as a function of conformation and/or simplifications of the representation of the polypeptide chain, such as one point per residue on a cubic lattice. It is not clear how the results of these theories are affected by their various simplifications. Here we take a very different simplification approach where the chain is accurately represented and the energy of each conformation is calculated by a not unreasonable empirical function. However, the set of amino acid sequences and allowed conformations is so restricted that it becomes computationally feasible to examine them all. Hence we are able to calculate melting curves for thermal denaturation as well as the detailed kinetic pathway of refolding. Such calculations are based on a novel representation of the conformations as points in an abstract 12-dimensional Euclidean conformation space. Fast folding sequences have relatively high melting temperatures, native structures with relatively low energies, small kinetic barriers between local minima, and relatively many conformations in the global energy minimum's watershed. In contrast to other folding theories, these models show no necessary relationship between fast folding and an overall funnel shape to the energy surface, or a large energy gap between the native and the lowest nonnative structure, or the depth of the native energy minimum compared to the roughness of the energy landscape. Proteins 32:425–437, 1998. © 1998 Wiley-Liss, Inc.     

The machinery of membrane protein assembly

The SecY (bacteria) and Sec61 (eukaryotes) translocon complexes, or protein-conducting channels, work in concert with bound ribosomes to insert proteins into membranes during the first step of membrane protein assembly. The crystallographic structure of an archaeal SecY translocon provides dramatic new insights into the mechanism of translocon function. This structure suggests an explanation for how the translocon can aid in establishing membrane protein topology via the positive-inside rule. The folding of membrane proteins may begin in the ribosome exit tunnel, before entering the translocon, according to cryo-electron microscopy and biophysical studies.     

Chromogranin, an integral membrane protein

Chromogranin is the major soluble protein of the adrenal medulla chromaffin granule and is secreted upon nervous stimulation. Using antisera to pure chromogranin in immunoblotting procedures, we show that chromogranin is the major integral membrane protein as well. Extraction of chromaffin granule membranes with low salt, high salt, chelating agents, or calcium-containing solutions does not remove the chromogranin from the membranes. The membrane form of chromogranin can be purified on a C-18 semi-preparative column using high pressure liquid chromatography. Amino-terminal sequence data indicate that the membrane and soluble forms of chromogranin are identical or very similar.     

Statistical mechanics of protein folding by cluster distance geometry

This is our second type of model for protein folding where the configurational parameters and the effective potential energy function are chosen in such a way that all conformations are described and the canonical partition function can be evaluated analytically. Structure is described in terms of distances between pairs of sequentially contiguous blocks of eight residues, and all possible conformations are grouped into 71 subsets in terms of bounds on these distances. The energy is taken to be a sum of pairwise interactions between such blocks. The 210 energy parameters were adjusted so that the native folds of 32 small proteins are favored in free energy over the denatured state. We then found 146 proteins having negligible sequence similarity to any of the training proteins, yet the free energy of the respective correct native states were favored over the denatured state.     

Statistical mechanics of protein folding with separable energy functions

We have initiated an entirely new approach to statistical mechanical models of strongly interacting systems where the configurational parameters and the potential energy function are both constructed so that the canonical partition function can be evaluated analytically. For a simplified model of proteins consisting of a single, fairly short polypeptide chain without cross-links, we can adjust the energy parameters to favor the experimentally determined native state of seven proteins having diverse types of folds. Then 497 test proteins are predicted to have stable native folds, even though they are also structurally diverse, and 480 of them have no significant sequence similarity to any of the training proteins.     

Electrostatic interactions in an integral membrane protein

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.     

Conformationally specific misfolding of an integral membrane protein

Membrane protein misfolding is related to the etiology of many diseases, but is poorly understood, particularly from a structural standpoint. This study focuses upon misfolding of a mutant form of diacylglycerol kinase (s-DAGK), a 40 kDa homotrimeric protein having nine transmembrane segments. Preparations of s-DAGK sometimes contain a kinetically trapped misfolded population, as evidenced by lower-than-expected enzyme activity (with no accompanying change in substrate K(m)) and by the appearance of a second band in electrophoresis gels. Misfolding of s-DAGK may take place during cellular overexpression, but can also be reproduced using the purified enzyme. TROSY NMR spectra of s-DAGK as a 100 kDa complex with detergent micelles exhibit a single additional set of resonances from the misfolded form, indicating a single misfolded conformational state. The relative intensities of these extra resonances correlate with the percent reduction in enzyme activity below the maximum observed for fully folded s-DAGK. Misfolded s-DAGK exhibits a modest difference in its far-UV CD spectrum compared to the folded enzyme, consistent with a small degree of variance in secondary structural content between the two forms. However, differences in NMR chemical shift dispersion and temperature-dependent line widths exhibited by folded and misfolded s-DAGK support the notion that they represent very different structural states. Cross-linking experiments indicate that both the correctly folded enzyme and the kinetically trapped misfolded form are homotrimers. This work appears to represent the first documentation of conformationally specific misfolding of an integral membrane protein.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Statistical mechanics of DNA and protein suitable for computer calculation

The metling behavior of DNA and formation of α-helices and β-strands in protein can be discussed rigorously by statistical mechanical treatment. To do this, however, appropriate formulations are required for fast computer calculation. New formalisms for DNA and protein in terms of recurrence relations suitable for computer calculation are presented.     

Rapid assembly of a multimeric membrane protein pore

We have observed the assembly of the staphylococcal pore-forming toxin α-hemolysin using single-molecule fluorescence imaging. Surprisingly, assembly from the monomer to the complete heptamer is extremely rapid, occurring in <5 ms. No lower order oligomeric intermediates are detected. Monte Carlo simulation of our experiment shows that assembly is diffusion limited, and pore formation is dependent on the stability of intermediate species. There are close similarities between bacterial pore-forming toxins, such as staphylococcal α-hemolysin, the anthrax protective antigen, and the cholesterol-dependent cytolysins, and their eukaryotic analogs, such as the complement pore membrane attack complex and perforin domain. The assembly mechanism we have observed for α-hemolysin provides a simple model that aids our understanding of these important pore formers.     

Characterization of the major integral protein of vacuolar membrane

The vacuolar membrane of radish (Raphanus sativus) taproot contained a large quantity of a protein of 23 kilodaltons that accounted for more than 25% of the total membrane proteins. The protein, tentatively named VM 23, was purified and characterized. VM 23 tends to aggregate at high temperature even in the presence of 1% sodium dodecyl sulfate. The apparent molecular size of VM 23 was estimated to be about 400 kilodaltons by polyacrylamide gel electrophoresis in the presence of 0.1% Triton X-100. VM 23 was partially extracted from the vacuolar membranes with chloroform:methanol, indicating its high hydrophobicity. The hydrophobic carboxyl modifier N,N′-dicyclohexylcarbodiimide bound covalently to VM 23. The results suggest that VM 23 may act as a secondary transport system coupled with the proton transport. The antibody against radish VM 23 reacted with the major proteins in the vacuolar membranes of mung bean (Vigna radiata) and castor bean (Ricinus communis) hypocotyls and pumpkin (Cucurbita moschata) epicotyl, but not with that of sugar beet (Beta vulgaris) taproot. VM 23 comigrated with vacuolar H⁺-pyrophosphatase on sucrose density gradient centrifugation after sonication of membranes, indicating that it is associated with the vacuolar membrane.     

Effect of membrane mechanics on charge transfer by the membrane protein prestin

Prestin was found in the membrane of outer hair cells (OHCs) located in the cochlea of the mammalian inner ear. These cells convert changes in the membrane potential into dimensional changes and (if constrained) to an active electromechanical force. The OHCs provide the ear with the mechanism of amplification and frequency selectivity that is effective up to tens of kHz. Prestin is a crucial part of the motor complex driving OHCs. Other cells transfected with prestin acquire electromechanical properties similar to those in the native cell. While the mechanism of prestin has yet to be fully understood, the charge transfer is its critical component. Here we investigate the effect of the mechanics of the surrounding membrane on electric charge transfer by prestin. We simulate changes in the membrane mechanics via the corresponding changes in the free energy of the prestin system. The free energy gradient enters a Fokker-Planck equation that describes charge transfer in our model. We analyze the effects of changes in the membrane tension and membrane elastic moduli. In the case of OHC, we simulate changes in the longitudinal and/or circumferential stiffness of the cell’s orthotropic composite membrane. In the case of cells transfected with prestin, we vary the membrane areal modulus. As a result, we show the effects of the membrane mechanics on the probabilistic characteristics of prestin-associated charge transfer for both stationary and high-frequency conditions. We compare our computational results with the available experimental data and find good agreement with the experiment.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

Stoichiometry of lipid-protein interaction and integral membrane protein structure

The stoichiometries of lipid-protein interaction obtained from spin label electron spin resonance experiments with integral membrane proteins are compared with simple geometric models for the intramembranous perimeter that are based on the predicted numbers of transmembrane helices. Deviations from the predicted values provide evidence for oligomerization of the protein in the membrane and/or more complex arrangements of the transmembrane segments. Received: 16 January 1997 / Accepted: 5 March 1997     

Targeting of the 22 kDa integral peroxisomal membrane protein

Investigating targeting of the 22 kDa peroxisomal membrane protein (Pmp22p) to the peroxisomal membrane we have confined the targeting signal to amino acid residues 16-37 located in the N-terminal cytoplasmic tail. Comparison of Pmp22p orthologous sequences revealed a conserved motif Y3xL3xP3x(KQN) which might represent the core of this targeting signal not found so far in other Pmps. Fusion of the Pmp22p N-terminal tail to the C-terminal portion of Pmp22p which per se is not targeted to peroxisomes, conveys peroxisomal targeting. These data suggest that Pmp22p is targeted to peroxisomes by a new membrane targeting signal which is necessary and sufficient to target a polypeptide containing two transmembrane spans to peroxisomes.