Biomedical subjects

Arthur E Johnson

Publications and source records attributed to Arthur E Johnson.

At least 19 recordsLinked to original sources

PubMed2006

The Tim9p/10p and Tim8p/13p complexes bind to specific sites on Tim23p during mitochondrial protein import.

The import of polytopic membrane proteins into the mitochondrial inner membrane (IM) is facilitated by Tim9p/Tim10p and Tim8p/Tim13p protein complexes in the intermembrane space (IMS). These complexes are proposed to act as chaperones by transporting the hydrophobic IM proteins through the aqueous IMS and preventing their aggregation. To examine the nature of this interaction, Tim23p molecules containing a single photoreactive cross-linking probe were imported into mitochondria in the absence of an IM potential where they associated with small Tim complexes in the IMS. On photolysis and immunoprecipitation, a probe located at a particular Tim23p site (27 different locations were examined) was found to react covalently with, in most cases, only one of the small Tim proteins. Tim8p, Tim9p, Tim10p, and Tim13p were therefore positioned adjacent to specific sites in the Tim23p substrate before its integration into the IM. This specificity of binding to Tim23p strongly suggests that small Tim proteins do not function solely as general chaperones by minimizing the exposure of nonpolar Tim23p surfaces to the aqueous medium, but may also align a folded Tim23p substrate in the proper orientation for delivery and integration into the IM at the TIM22 translocon.

Binding Sites↗

PubMed2006

Sec61alpha and TRAM are sequentially adjacent to a nascent viral membrane protein during its ER integration.

Co-translational integration of a nascent viral membrane protein into the endoplasmic reticulum membrane takes place via the translocon. We have been studying the early stages of the integration of a double-spanning plant viral movement protein to gain insights into how viral membrane proteins are transferred from the hydrophilic interior of the translocon into the hydrophobic environment of the bilayer, where the transmembrane (TM) segments of the viral proteins can diffuse freely. Photocrosslinking experiments reveal that this integration involves the sequential passage of the TM segments past Sec61alpha and translocating chain-associating membrane protein (TRAM). Each TM segment is first adjacent to Sec61alpha and subsequently is adjacent to TRAM. TRAM crosslinking extends for a long period during nascent chain biogenesis. In addition, the replacement of the first viral TM segment with a non-viral TM sequence still yields nascent chain photo-adducts with TRAM. TRAM therefore appears to be involved in viral membrane protein integration, and nascent chain recognition by TRAM does not appear to rely solely on the TM domains.

Animals↗

PubMed2006

Translation arrest requires two-way communication between a nascent polypeptide and the ribosome.

When the export of E. coli SecM is blocked, a 17 amino acid motif near the C terminus of the protein induces a translation arrest from within the ribosome tunnel. Here we used a recently described application of fluorescence resonance energy transfer (FRET) to gain insight into the mechanism of translation arrest. We found that the SecM C terminus adopted a compact conformation upon synthesis of the arrest motif. This conformational change did not occur spontaneously, but rather was induced by the ribosome. Translation arrest required both compaction of the SecM C terminus and the presence of key residues in the arrest motif. Further analysis showed that the arrested peptidyl-tRNA was resistant to puromycin treatment and revealed additional changes in the ribosome-nascent SecM complex. Based on these observations, we propose that translation arrest results from a series of reciprocal interactions between the ribosome and the C terminus of the nascent SecM polypeptide.

Amino Acid Motifs↗

PubMed2006

Importin-alpha-16 is a translocon-associated protein involved in sorting membrane proteins to the nuclear envelope.

A viral inner nuclear membrane-sorting motif sequence (INM-SM) was used to identify proteins that recognize integral membrane proteins destined for the INM. Herein we describe importin-alpha-16, a membrane-associated isoform of Spodoptera frugiperda importin-alpha that contains the C-terminal amino acid residues comprising armadillo helical-repeat domains 7-10. In the endoplasmic reticulum (ER) membrane, importin-alpha-16 is adjacent to the translocon protein Sec61alpha. Importin-alpha-16 cross-links to the INM-SM sequence as it emerges from the ribosomal tunnel and remains adjacent to the INM-SM after INM-SM integration into the ER membrane and release from the translocon. Cross-linking results suggest that importin-alpha-16 discriminates between INM- and non-INM-directed proteins. Thus, it seems that during and after cotranslational membrane integration, importin-alpha-16 is involved in the trafficking of integral membrane proteins to the INM.

Amino Acid Sequence↗

PubMed2006

Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids.

Synaptotagmin acts as a Ca(2+) sensor in neurotransmitter release through its two C(2) domains. Ca(2+)-dependent phospholipid binding is key for synaptotagmin function, but it is unclear how this activity cooperates with the SNARE complex involved in release or why Ca(2+) binding to the C(2)B domain is more crucial for release than Ca(2+) binding to the C(2)A domain. Here we show that Ca(2+) induces high-affinity simultaneous binding of synaptotagmin to two membranes, bringing them into close proximity. The synaptotagmin C(2)B domain is sufficient for this ability, which arises from the abundance of basic residues around its surface. We propose a model wherein synaptotagmin cooperates with the SNAREs in bringing the synaptic vesicle and plasma membranes together and accelerates membrane fusion through the highly positive electrostatic potential of its C(2)B domain.

Animals↗

PubMed2006

Docosahexaenoic acid selectively inhibits plasma membrane targeting of lipidated proteins.

Membrane localization of lipidated cytosolic signaling proteins is mediated by interactions between specific lipid anchors and membranes, but little is known about the regulatory role of membrane composition in lipidated protein membrane targeting. Here, using green fluorescent protein (GFP) chimeras and quantitative fluorescence microscopy in living mouse colonocytes, we show that docosahexaenoic acid (DHA), a dietary polyunsaturated fatty acid (PUFA) with membrane lipid-modifying properties, selectively inhibits plasma membrane (PM) targeting and increases the endomembrane localization of lipidated proteins that are cytoplasmic cargo in the exocytic pathway, without affecting the exocytic pathway itself. DHA selectivity seems to be dictated by the protein trafficking route, independent of the functional state of proteins and the location and composition of membrane anchors. DHA enrichment in cell membranes was required to elicit the inhibitory effect. These data reveal that membrane lipid composition influences cell signaling by modulating intracellular trafficking and localization of membrane proteins, providing a potential molecular mechanism for the documented health benefits of DHA.

Animals↗

PubMed2005

Exploring the capacity of trigger factor to function as a shield for ribosome bound polypeptide chains.

Ribosome-bound trigger factor (TF) is the first chaperone encountered by a nascent polypeptide chain in bacteria. TF has been proposed to form a cradle-shaped shield for nascent chains up to approximately 130 residues to fold in a protected environment upon exit from the ribosome. We report that nascent chains of luciferase up to 280 residues in length are relatively protected by TF against digestion by proteinase K. In contrast, nascent chains of the constitutively unstructured protein alpha-synuclein were not protected, although they were in close proximity to TF by crosslinking. Thus, TF is not a general shield for nascent chains. Protease protection appears to depend on a hydrophobic interaction of TF with nascent polypeptides.

Animals↗

PubMed2005

Mapping the interaction of the STT3 subunit of the oligosaccharyl transferase complex with nascent polypeptide chains.

Many secretory and membrane proteins are N-glycosylated by the oligosaccharyl transferase complex during their translocation across the endoplasmic reticulum membrane. Several experimental observations suggest that the highly conserved STT3 subunit contains the active site of the oligosaccharyl transferase. Here, we report a detailed study of the interaction between the active site of the STT3 protein and nascent polypeptide chains using an in vitro photocrosslinking technique. Our results show that the addition of a glycan moiety in a stretch of approximately 15 residues surrounding a QK(*)T cross-linking site impairs the interaction between the nascent chain and STT3.

Animals↗

PubMed2005

Sequential triage of transmembrane segments by Sec61alpha during biogenesis of a native multispanning membrane protein.

During polytopic protein biogenesis, the Sec61 translocon must rapidly orient and integrate multiple transmembrane segments (TMs) into the endoplasmic reticulum membrane. To understand this process, we examined interactions between Sec61alpha and all six TMs of the aquaporin-4 (AQP4) water channel at defined stages of synthesis using incorporated photo-cross-linking probes. Each TM interacted with and moved through the translocon in a highly ordered and sequential fashion. Strong asymmetric Sec61alpha cross-linking was observed for only one helix at a time, suggesting the presence of a single primary binding site. However, up to four TMs simultaneously contacted Sec61alpha from different molecular environments. Thus, AQP4 integration by Sec61alpha involves sequential triage of TMs from their initial portal of entry into multiple secondary sites within the translocon. This mechanism provides a means to facilitate early folding events before release into the lipid bilayer.

Animals↗

PubMed2005

Selective SecA association with signal sequences in ribosome-bound nascent chains: a potential role for SecA in ribosome targeting to the bacterial membrane.

The role of SecA in selecting bacterial proteins for export was examined using a heterologous system that lacks endogenous SecA and other bacterial proteins. This approach allowed us to assess the interaction of SecA with ribosome-bound photoreactive nascent chains in the absence of trigger factor, SecB, Ffh (the bacterial protein component of the signal recognition particle), and the SecYEG translocon in the bacterial plasma membrane. In the absence of membranes, SecA photocross-linked efficiently to nascent translocation substrate OmpA in ribosome-nascent chain (RNC) complexes in an interaction that was independent of both ATP and SecB. However, no photocross-linking to a nascent membrane protein that is normally targeted by a signal recognition particle was observed. Modification of the signal sequence revealed that its affinity for SecA and Ffh varied inversely. Gel filtration showed that SecA binds tightly to both translating and non-translating ribosomes. When purified SecA.RNC complexes containing nascent OmpA were exposed to inner membrane vesicles lacking functional SecA, the nascent chains were successfully targeted to SecYEG translocons. However, purified RNCs lacking SecA were unable to target to the same membranes. Taken together, these data strongly suggest that cytosolic SecA participates in the selection of proteins for export by co-translationally binding to the signal sequences of non-membrane proteins and directing those nascent chains to the translocon.

Adenosine Triphosphatases↗

PubMed2005

The cotranslational contacts between ribosome-bound nascent polypeptides and the subunits of the hetero-oligomeric chaperonin TRiC probed by photocross-linking.

The hetero-oligomeric eukaryotic chaperonin TRiC (TCP-1-ring complex, also called CCT) interacts cotranslationally with a diverse subset of newly synthesized proteins, including actin, tubulin, and luciferase, and facilitates their correct folding. A photocross-linking approach has been used to map the contacts between individual chaperonin subunits and ribosome-bound nascent chains of increasing length. Whereas a cryo-EM study suggests that chemically denatured actin interacts with only two TRiC subunits (delta and either beta or epsilon), actin and luciferase chains photocross-link to at least six TRiC subunits (alpha, beta, delta, epsilon, xi, and theta) at different stages of translation. Furthermore, the photocross-linking of actin, but not luciferase, nascent chains to TRiC subunits zeta and theta was length-dependent. In addition, a single photoreactive probe incorporated at a unique site in actin nascent chains of different lengths reacted covalently with multiple TRiC subunits, thereby indicating that the nascent chain samples the polypeptide binding sites of different subunits. We conclude that elongating actin and luciferase nascent chains contact multiple TRiC subunits upon emerging from the ribosome, and that the TRiC subunits contacted by nascent actin change as it elongates and starts to fold.

Actins↗

PubMed2005

Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted.

The current model for cell-to-cell movement of plant viruses holds that transport requires virus-encoded movement proteins that intimately associate with endoplasmic reticulum membranes. We have examined the early stages of the integration into endoplasmic reticulum membranes of a double-spanning viral movement protein using photocross-linking. We have discovered that this process is cotranslational and proceeds in a signal recognition particle-dependent manner. In addition, nascent chain photocross-linking to Sec61alpha and translocating chain-associated membrane protein reveal that viral membrane protein insertion takes place via the translocon, as with most eukaryotic membrane proteins, but that the two transmembrane segments of the viral protein leave the translocon and enter the lipid bilayer together.

Calcium-Binding Proteins↗

PubMed2005

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation.

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.

Bacterial Toxins↗

PubMed2005

The co-translational folding and interactions of nascent protein chains: a new approach using fluorescence resonance energy transfer.

During protein biosynthesis, a nascent protein is exposed to multiple environments and proteins both inside and outside the ribosome that influence nascent chain folding and trafficking. Fluorescence resonance energy transfer between two dyes incorporated into a single nascent chain using aminoacyl-tRNA analogs can directly and selectively monitor changes in nascent chain conformation. This approach recently revealed the existence and functional ramifications of ribosome-mediated folding of nascent membrane proteins inside the ribosome and can be extended to characterize the effects of chaperones and other proteins and ligands on nascent protein folding, interactions, assembly, and avoidance of misfolding and degradation.

Fluorescence Resonance Energy Transfer↗

PubMed2005

The molecular mechanisms underlying BiP-mediated gating of the Sec61 translocon of the endoplasmic reticulum.

The Sec61 translocon of the endoplasmic reticulum membrane forms an aqueous pore that is gated by the lumenal Hsp70 chaperone BiP. We have explored the molecular mechanisms governing BiP-mediated gating activity, including the coupling between gating and the BiP ATPase cycle, and the involvement of the substrate-binding and J domain-binding regions of BiP. Translocon gating was assayed by measuring the collisional quenching of fluorescent probes incorporated into nascent chains of translocation intermediates engaged with microsomes containing various BiP mutants and BiP substrate. Our results indicate that BiP must assume the ADP-bound conformation to seal the translocon, and that the reopening of the pore requires an ATP binding-induced conformational change. Further, pore closure requires functional interactions between both the substrate-binding region and the J domain-binding region of BiP and membrane proteins. The mechanism by which BiP mediates translocon pore closure and opening is therefore similar to that in which Hsp70 chaperones associate with and dissociate from substrates.

Adenosine Diphosphate↗

PubMed2005

Fluorescence approaches for determining protein conformations, interactions and mechanisms at membranes.

Processes that occur at membranes are essential for the viability of every cell, but such processes are the least well understood at the molecular level. The complex nature and physical properties of the molecular components involved, as well as the requirement for two separated aqueous compartments, restrict the experimental approaches that can be successfully applied to examine the structure, conformational changes and interactions of the membrane-bound proteins that accomplish these processes. In particular, to accurately elucidate the molecular mechanisms that effect and regulate such processes, one must use experimental approaches that do not disrupt the structural integrity or functionality of the protein-membrane complexes being examined. To best accomplish this goal, especially when large multicomponent complexes and native membranes are involved, the optimal experimental approach to use is most often fluorescence spectroscopy. Using multiple independent fluorescence techniques, one can determine structural information in real time and in intact membranes under native conditions that cannot be obtained by crystallography, electron microscopy and NMR techniques, among others. Furthermore, fluorescence techniques provide a comprehensive range of information, from kinetic to thermodynamic, about the assembly, structure, function and regulation of membrane-bound proteins and complexes. This article describes the use of various fluorescence techniques to characterize different aspects of proteins bound to or embedded in membranes.

Animals↗

PubMed2004

Conserved tertiary base pairing ensures proper RNA folding and efficient assembly of the signal recognition particle Alu domain.

Proper folding of the RNA is an essential step in the assembly of functional ribonucleoprotein complexes. We examined the role of conserved base pairs formed between two distant loops in the Alu portion of the mammalian signal recognition particle RNA (SRP RNA) in SRP assembly and functions. Mutations disrupting base pairing interfere with folding of the Alu portion of the SRP RNA as monitored by probing the RNA structure and the binding of the protein SRP9/14. Complementary mutations rescue the defect establishing a role of the tertiary loop-loop interaction in RNA folding. The same mutations in the Alu domain have no major effect on binding of proteins to the S domain suggesting that the S domain can fold independently. Once assembled into a complete SRP, even particles that contain mutant RNA are active in arresting nascent chain elongation and translocation into microsomes, and, therefore, tertiary base pairing does not appear to be essential for these activities. Our results suggest a model in which the loop-loop interaction and binding of the protein SRP9/14 play an important role in the early steps of SRP RNA folding and assembly.

Alu Elements↗

PubMed2004

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane.

The current diffusion-retention model for protein trafficking to the inner nuclear membrane (INM) proposes that INM proteins diffuse laterally from the membrane of the endoplasmic reticulum into the INM and are then retained in the INM by binding to nuclear proteins or DNA. Because some data indicate that the sorting of baculovirus envelope proteins to the INM is protein-mediated, we have examined the early stages of INM protein integration and sorting by using photocrosslinking. Both viral and host INM-directed proteins were integrated cotranslationally through the endoplasmic reticulum translocon, and their nonrandom photocrosslinking to two translocon proteins, Sec61alpha and translocating chain-associated membrane protein (TRAM), revealed that the first transmembrane sequence (TMS) of each viral and host INM-directed protein occupied a very similar location within the translocon. Because few TMSs of non-INM-directed membrane proteins photocrosslink to TRAM, it seems that the INM-directed TMSs occupy different sites within the translocon than do non-INM-directed TMSs. The distinct proximities of translocon components to INM-directed TMSs strongly suggest that such TMSs are recognized and initially sorted within the translocon. Taken together, these data indicate that membrane protein sorting to the INM is an active process involving specific nonnuclear proteins.

Amino Acid Sequence↗