A general mechanism for regulation of access to the translocon: Competition for a membrane attachment site on ribosomes

Möller, Ines; Jung, Martin; Beatrix, Birgitta; Levy, Robert J.; Kreibich, Gert; Zimmermann, Richard; Wiedmann, Martin; Lauring, Brett

doi:10.1073/pnas.95.23.13425

Cited by 68 publications

(55 citation statements)

References 31 publications

(39 reference statements)

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“…Eukaryotic NAC is an important protein complex functioning as a stable heterodimer of αNAC and βNAC subunits in cotranslation targeting (Lauring et al, 1995a, b;Powers and Walter, 1996;Moller et al, 1998b) and X-junction related pathways (Whitby and Dixon, 2001) among others. The crystal structure of homologous aeNAC from archaea, which has only one NAC-like gene, shows that aeNAC forms a homodimer through the NAC domains (Spreter et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

“…The binding and releasing of NAC for nascent polypeptide appear in quantal units instead of a residue-byresidue manner. This binding is independent of signal peptide sequences, and yet NAC cooperates with signal recognition particle (SRP) for correctly targeting and folding of nascent polypeptide in the cotranslational process (Lauring et al, 1995a, b;Powers and Walter, 1996;Moller et al, 1998b). However, yeast NAC homolog (termed as EGD complex) is not prerequisite for growth (Reimann et al, 1999), but is necessary for protein targeting to mitochondria (George et al, 1998;Fünfschilling and Rospert, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

et al. 2010

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Nascent polypeptide associated complex (NAC) and its two isolated subunits, αNAC and βNAC, play important roles in nascent peptide targeting. We determined a 1.9 Å resolution crystal structure of the interaction core of NAC heterodimer and a 2.4 Å resolution crystal structure of αNAC NAC domain homodimer. These structures provide detailed information of NAC heterodimerization and αNAC homodimerization. We found that the NAC domains of αNAC and βNAC share very similar folding despite of their relative low identity of amino acid sequences. Furthermore, different electric charge distributions of the two subunits at the NAC interface provide an explanation to the observation that the heterodimer of NAC complex is more stable than the single subunit homodimer. In addition, we successfully built a βNAC NAC domain homodimer model based on homologous modeling, suggesting that NAC domain dimerization is a general property of the NAC family. These 3D structures allow further studies on structurefunction relationship of NAC.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

et al. 2010

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“…The technology, developed by investigators primarily interested in protein translocation across membranes, has been successfully utilized to identify proteins comprising the translocation pores in ER (45,51) and mitochondrial membranes (52). In addition, it has been useful in tracing the temporal changes in molecular interactions that newly synthesized proteins undergo as they execute their folding program (53)(54)(55).…”

Section: Resultsmentioning

confidence: 99%

Parkinson's Disease-associated α-Synuclein Is a Calmodulin Substrate

Martinez

Moeller

Erdjument‐Bromage

et al. 2003

Journal of Biological Chemistry

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␣-Synuclein is a neuronal protein thought to be central in the pathogenesis of Parkinson's disease (PD) because it comprises the fibrillar core of Lewy bodies, one of the histologically defining lesions of PD, and because mutations in ␣-synuclein cause autosomal dominant PD. Although its physiologic role is uncertain, ␣-synuclein is a synaptic protein that may contribute to plasticity. We produced synuclein with incorporated photoprobes to identify and purify novel synuclein-interacting proteins both to begin to clarify the physiology of synuclein and to identify factors that may regulate synuclein conformation. We detected several cross-links and purified and identified one as calmodulin (CaM). CaM binds to both wild type and PD-associated mutant ␣-synucleins in a calcium-dependent manner. We further demonstrate that CaM and ␣-synuclein interact in intact cells in a calcium-dependent manner and that activated CaM accelerates the formation of synuclein fibrils in vitro. We hypothesize that the known calcium control of synuclein function is mediated through CaM interaction and that CaM potentially alters synuclein conformation.

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“…those lacking a signal peptide (7). NAC also prevents binding of inappropriate ribosomes to translocons on the ER by blocking the putative ribosomal M-site (1,10,23,26). We have previously hypothesized that, as the nascent chain lengthens, NAC binding weakens, and for signal sequence containing proteins, allows SRP to compete effectively for binding to signal peptides.…”

Section: Discussionmentioning

confidence: 99%

“…␤NAC Alone Prevents Default Targeting of Ribosomes Containing Signal-less Nascent Chains to the ER-In addition to its role in shielding the nascent chain on the ribosome toward the cytosol, NAC prevents inappropriate targeting of nontranslating ribosomes and RNCs bearing signal-less nascent chains to the ER membrane by occupying the proposed membrane-attachment site (M-site) on the ribosome (23,26). We next investigated whether the binding of ␤NAC and recNAC to ribosomes observed in Fig.…”

Section: Fig 4 Recombinant Nac Interacts With Nascent Chains On Ribmentioning

confidence: 99%

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

Beatrix

Sakai

Wiedmann

2000

Journal of Biological Chemistry

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Nascent polypeptide-associated complex (NAC) is probably the first cytosolic protein to contact nascent polypeptide chains emerging from ribosomes. In this way NAC prevents inappropriate interactions with other factors. Eventually other factors involved in targeting and folding, like the Signal Recognition Particle or cytosolic chaperones, must gain access to the nascent chain. All NAC preparations to date consist of two copurifying polypeptides. Here we rigorously show that these two polypeptides, termed ␣-and ␤NAC, form a very stable complex in vivo and in vitro and that a functional complex can be reconstituted from the individual subunits. A dissection of the contributions of the individual subunits to NACs function revealed that both subunits are in direct contact with nascent polypeptide chains on the ribosome and that both contribute to the prevention of inappropriate interactions. However, ␤NAC alone directly binds to the ribosome and is sufficient to prevent ribosome binding to the endoplasmic reticulum membrane. Nascent polypeptide-associated complex (NAC) 1 is a very abundant cytosolic protein, which is involved in cotranslational targeting of polypeptides to the endoplasmic reticulum (ER) membrane. The intracellular NAC concentration varies only slightly in different tissues, ranging from 3 to 10 M (1). NAC is highly conserved among eukaryotes, but up to now no functional prokaryotic homolog has been described. The importance of NACs in vivo function is emphasized by early embryonically lethal phenotypes of NAC mutants in mice and fruit flies (2, 3). Recently it was also shown that yeast NAC, although not essential for growth (4), is involved in the import of proteins into mitochondria (5, 6).NAC was originally identified as a ribosome-associated protein when we set out to probe the molecular environment of growing polypeptides on the ribosome using a photo-cross-linking approach (7). With this technique we were able to show that NAC can interact with regions of nascent chains as close as 17 amino acids to the peptidyl transferase center. Additionally, it was shown by protease protection that NAC functions as a dissociable wall of a ribosomal tunnel through which the growing polypeptide emerges (8). Cycles of binding and releasing NAC expose the polypeptide to the cytosol in "quantal units" rather than amino acid by amino acid. We have proposed that exposing the polypeptide chain in functional units contributes to fidelity in cotranslational processes such as targeting and folding (9). Depleting NAC from translating ribosomes led to at least two inappropriate interactions. First, the signal recognition particle (SRP), which interacts with signal peptides of nascent chains on ribosomes during targeting to the ER membrane, could be cross-linked to nascent chains lacking signal peptides (7). Second, in the absence of NAC ribosomes translating non-secretory polypeptides inappropriately interacted with translocation sites on ER membranes and the signal-less chains were even translocated, although with l...

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A general mechanism for regulation of access to the translocon: Competition for a membrane attachment site on ribosomes

Cited by 68 publications

References 31 publications

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Crystal structures of NAC domains of human nascent polypeptide-associated complex (NAC) and its αNAC subunit

Parkinson's Disease-associated α-Synuclein Is a Calmodulin Substrate

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

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