The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.

em alguns aspectos, a presente invencao proporciona uma composicao de nanoparticulas compreendendo rnat e um composto de entrega aminolipidico. o composto de entrega aminolipidico pode ser um dendrimero, dendron ou lipideo dendritico, um polimero, tal como uma poliamida ou poliester, ou um lipideo com um ou mais componentes hidrofobicos. em algumas formas de realizacao, estas composicoes podem ser administradas a um paciente para tratar uma doenca ou disturbio genetico, tal como a fibrose cistica, distrofia muscular de duchene ou câncer.

The majority of cholesterol-dependent cytolysins (CDCs) utilize cholesterol as a membrane receptor, whereas a small number are restricted to the GPI-anchored protein CD59 for initial membrane recognition. Two cholesterol-binding CDCs, perfringolysin O (PFO) and streptolysin O (SLO), were found to exhibit strikingly different binding properties to cholesterol-rich natural and synthetic membranes. The structural basis for this difference was mapped to one of the loops (L3) in the membrane binding interface that help anchor the toxin monomers to the membrane after receptor (cholesterol) binding by the membrane insertion of its amino acid side chains. A single point mutation in this loop conferred the binding properties of SLO to PFO and vice versa. Our studies strongly suggest that changing the side chain structure of this loop alters its equilibrium between membrane-inserted and uninserted states, thereby affecting the overall binding affinity and total bound toxin. Previous studies have shown that the lipid environment of cholesterol has a dramatic effect on binding and activity. Combining this data with the results of our current studies on L3 suggests that the structure of this loop has evolved in the different CDCs to preferentially direct binding to cholesterol in different lipid environments. Finally, the efficiency of β-barrel pore formation was inversely correlated with the increased binding and affinity of the PFO L3 mutant, suggesting that selection of a compatible lipid environment impacts the efficiency of membrane insertion of the β-barrel pore.

The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about NPHP4's function, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. A nphp4 null mutant forms flagella with nearly normal length, ultrastructure, and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but a subset of membrane proteins was greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the TZ barrier.

The TIM23 Complex of the mitochondrial inner membrane (IM) is a multi-component assembly that mediates the translocation of matrix-targeted precursor proteins as well as the integration of membrane proteins. This complex is energetically coupled to both an ATPase motor and the electrochemical proton potential across the IM. The central subunit of this complex, Tim23, forms a voltage-gated channel and contains a large soluble domain in the intermembrane space that functions as a substrate receptor. To analyze the structural dynamics that are attendant with changes in the energized state of the membrane, we employed a high-resolution fluorescence mapping approach in which small extrinsic probes were cotranslationally incorporated into specific sites of Tim23 from Saccharomyces cerevisiae and analyzed in active mitochondria by steady-state and time-resolved measurements under different physiologically relevant states. Analysis of the channel-facing transmembrane segment (TMS2) of Tim23 revealed that changes in the energized status of the inner membrane caused dramatic structural alterations in the channel region. In an energized membrane, TMS2 formed a continuous α-helix that was inaccessible to the aqueous intermembrane space. Upon depolarization, the helical periodicity of TMS2 was disrupted and the channel became exposed to the IMS. Real time kinetic measurements confirmed that changes in TMS2 conformation coincided with depolarization. This analysis is extended to the soluble receptor domain of Tim23, where we show proton-motive force-coupled structural changes and key protein interactions that are mediated by specific lipids within the inner membrane. These results reveal how the energized state of the membrane drives functionally relevant structural dynamics in membrane proteins that are coupled to processes such as channel gating.

Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP).

The assembly of the cholesterol-dependent cytolysin (CDC) oligomeric pore complex requires a complex choreography of secondary and tertiary structural changes in domain 3 (D3) of the CDC monomer structure. A point mutation was identified in the archetype CDC, perfringolysin O, that blocks detectable D3 structural changes and traps the membrane-bound monomers in an early and reversible stage of oligomer assembly. Using this and other mutants we show that specific D3 structural changes are propagated from one membrane-bound monomer to another. Propagation of these structural changes results in the exposure of a β-strand in D3 that allows it to pair and form edge-on interactions with a second β-strand of a free membrane-bound monomer. Pairing of these strands establishes the final geometry of the pore complex and is necessary to drive the formation of the β-barrel pore. These studies provide new insights into how structural information is propagated between membrane-bound monomers of a self-assembling system and the interactions that establish the geometry of the final pore complex.

The Rockefeller University Press $30.00 J. Cell Biol. Vol. 195 No. 1 41–54 www.jcb.org/cgi/doi/10.1083/jcb.201103117 JCB 41 P.-J. Lin and C.G. Jongsma contributed equally to this paper. Correspondence to Arthur E. Johnson: ajohnson@medicine.tamhsc.edu Abbreviations used in this paper: BiP, binding immunoglobulin protein; NBDLys, N-6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoyl-Lys; MLT, melittin; PMP, polytopic membrane protein; pPL, preprolactin; PTC, peptidyl transferase center; RNC, ribosome-nascent chain complex; RTC, ribosome–translocon complex; SSMP, single-spanning membrane protein; TMS, transmembrane segment; VSVG, vesicular stomatitis virus protein G. Introduction

Induction of ovarian activity and ovulation is a valuable tool for the genetic management of ex situ populations of wildlife. Deslorelin, a gonadotropin releasing hormone (GnRH)-agonist, has been used earlier to induce oestrus in the grey wolf (Canis lupus). The objective of the present study was to determine the efficacy of Deslorelin (2.1 mg, Ovuplant®, Peptech Animal Health, Australia) for manipulating ovarian activity of the maned wolf, a species speculated to be an induced ovulator. Eight female (4–12 years old) maned wolves were (i) paired with a male (n = 3) or (ii) housed alone (n = 5). Of the 8 females, 1 (1 in a pair and 1 singleton) were implanted with Deslorelin under the vulvar mucosa for 12 days. The remaining 6 received implants in the subcutaneous layer of the ear for 7 days. Fresh fecal samples were collected 5 to 7 days/week for 1 month before Deslorelin treatment through 6 weeks after implant withdrawal. Reproductive steroid metabolites were extracted from the fecal samples and quantified using a validated enzyme immunoassay. Baseline progestagen concentrations for each individual were calculated by an iterative process, whereby high values exceeding the mean plus one standard deviation were excluded. Comparisons of oestrogen and progestagen concentrations among pre-, peri-, and post-Deslorelin implant periods were performed using analysis of variance. Site and duration of the GnRH agonist treatment had no effect (P > 0.05) on subsequent ovarian responses. In paired females, oestrogen metabolites reached the highest (P < 0.05) concentration during Deslorelin (i.e. peri-) treatment (441.7 ± 20.2 ng g–1 of feces) compared to pre- (174.9 ± 16.7) and post- (177.8 ± 9.1) treatment. Progesterone metabolites rose (P < 0.05) above the baseline (indicative of ovulation) starting on Day 10 after the onset of Deslorelin implantation and remained elevated throughout the study (pre-, 11 645 ± 4798; peri-, 31 521 ± 6444; post-, 55 843 ± 2924 ng g–1 of feces). In singleton females, oestrogen metabolites increased (P < 0.05) immediately after implantation (from pre-, 184.2 ± 45.3 to peri-, 334.2 ± 29.8 ng g–1 of feces) and then declined (post-, 192.3 ± 12.4). Progestagen metabolites exhibited a similar pattern with a rise (P < 0.05) during Deslorelin treatment (from pre-, 3870 ± 1336 to peri-, 10 546 ± 880 ng g–1 of feces) followed by a decline after implant withdrawal (post-, 6171 ± 366), indicating that ovulation did not occur. These results suggest that Deslorelin can induce ovarian activity in the maned wolf. However, administration of an ovulatory agent after Deslorelin treatment may be required in females managed in the absence of a male, further supporting the assertion that this species is an induced ovulator. Funded by the Morris Animal Foundation.

In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility, and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.

The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (FRET) to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389-500 emerge from the ribosome exit tunnel, initiating compaction of a small, N-terminal α/β-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

Multi-spanning membrane protein loops are directed alternately into the cytosol or ER lumen during cotranslational integration. Nascent chain exposure is switched after a newly synthesized transmembrane segment (TMS) enters the ribosomal tunnel. FRET measurements revealed that each TMS is initially extended, but folds into a compact conformation after moving 6-7 residues from the peptidyltransferase center, irrespective of loop size. The ribosome-induced folding of each TMS coincided with its photocrosslinking to ribosomal protein L17 and an inversion of compartmental exposure. This correlation indicates that successive TMSs fold and bind at a specific ribosomal tunnel site that includes L17, thereby triggering structural rearrangements of multiple components in and on both sides of the ER membrane, most likely via TMS-dependent L17 and/or rRNA conformational changes transmitted to the surface. Thus, cyclical changes at the membrane during integration are initiated by TMS folding, even though nascent chain conformation and location vary dynamically in the ribosome tunnel. Nascent chains therefore control their own trafficking.

ABSTRACT Plant viral infection and spread depends on the successful introduction of a virus into a cell of a compatible host, followed by replication and cell-to-cell transport. The movement proteins (MPs) p8 and p9 of Turnip crinkle virus are required for cell-to-cell movement of the virus. We have examined the membrane association of p9 and found that it is an integral membrane protein with a defined topology in the endoplasmic reticulum (ER) membrane. Furthermore, we have used a site-specific photo-cross-linking strategy to study the membrane integration of the protein at the initial stages of its biosynthetic process. This process is cotranslational and proceeds through the signal recognition particle and the translocon complex.

The recognition and binding of cholesterol is an important feature of many eukaryotic, viral, and prokaryotic proteins, but the molecular details of such interactions are understood only for a few proteins. The pore-forming cholesterol-dependent cytolysins (CDCs) contribute to the pathogenic mechanisms of a large number of Gram-positive bacteria. Cholesterol dependence of the CDC mechanism is a hallmark of these toxins, yet the identity of the CDC cholesterol recognition motif has remained elusive. A detailed analysis of membrane interactive structures at the tip of perfringolysin O (PFO) domain 4 reveals that a threonine-leucine pair mediates CDC recognition of and binding to membrane cholesterol. This motif is conserved in all known CDCs and conservative changes in its sequence or order are not well tolerated. Thus, the Thr-Leu pair constitutes a common structural basis for mediating CDC-cholesterol recognition and binding, and defines a unique paradigm for membrane cholesterol recognition by surface-binding proteins.

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1-3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1-3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1-3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.