Abstract Protein biogenesis is essential in all cells and initiates when a nascent polypeptide emerges from the ribosome exit tunnel, where multiple ribosome-associated protein biogenesis factors (RPBs) direct nascent proteins to distinct fates. How distinct RPBs spatiotemporally coordinate with one another to affect accurate protein biogenesis is an emerging question. Here, we address this question by studying the role of a cotranslational chaperone, nascent polypeptide-associated complex (NAC), in regulating substrate selection by signal recognition particle (SRP), a universally conserved protein targeting machine. We show that mammalian SRP and SRP receptors (SR) are insufficient to generate the biologically required specificity for protein targeting to the endoplasmic reticulum. NAC co-binds with and remodels the conformational landscape of SRP on the ribosome to regulate its interaction kinetics with SR, thereby reducing the nonspecific targeting of signalless ribosomes and pre-emptive targeting of ribosomes with short nascent chains. Mathematical modeling demonstrates that the NAC-induced regulations of SRP activity are essential for the fidelity of cotranslational protein targeting. Our work establishes a molecular model for how NAC acts as a triage factor to prevent protein mislocalization, and demonstrates how the macromolecular crowding of RPBs at the ribosome exit site enhances the fidelity of substrate selection into individual protein biogenesis pathways.
more »
« less
Fidelity of Cotranslational Protein Targeting to the Endoplasmic Reticulum
Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.
more »
« less
- Award ID(s):
- 1929452
- PAR ID:
- 10354898
- Date Published:
- Journal Name:
- International Journal of Molecular Sciences
- Volume:
- 23
- Issue:
- 1
- ISSN:
- 1422-0067
- Page Range / eLocation ID:
- 281
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.more » « less
-
The conserved signal recognition particle (SRP) cotranslationally delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum (ER). The molecular mechanism by which eukaryotic SRP transitions from cargo recognition in the cytosol to protein translocation at the ER is not understood. Here, structural, biochemical, and single-molecule studies show that this transition requires multiple sequential conformational rearrangements in the targeting complex initiated by guanosine triphosphatase (GTPase)–driven compaction of the SRP receptor (SR). Disruption of these rearrangements, particularly in mutant SRP54 G226E linked to severe congenital neutropenia, uncouples the SRP/SR GTPase cycle from protein translocation. Structures of targeting intermediates reveal the molecular basis of early SRP-SR recognition and emphasize the role of eukaryote-specific elements in regulating targeting. Our results provide a molecular model for the structural and functional transitions of SRP throughout the targeting cycle and show that these transitions provide important points for biological regulation that can be perturbed in genetic diseases.more » « less
-
ABSTRACT Specialized membrane and cortical protein regions are common features of cells and are utilized to isolate differential cellular functions. In Drosophila photoreceptors, the apical membrane domain is defined by two distinct morphological membranes: the rhabdomere microvilli and the stalk membrane. To define the apical cortical protein complexes, we performed proximity labeling screens utilizing the rhabdomeric-specific protein PIP82 as bait. We found that the PIP82 interactome is enriched in actin-binding and cytoskeleton proteins, as well as proteins for cellular trafficking. Analysis of one target, Bifocal, with PIP82 revealed two independent pathways for localization to the rhabdomeric membrane and an additional mechanism of crosstalk between the protein complexes of the rhabdomeric and stalk membranes. The loss of Bifocal, and enhancement in the PIP82, bifocal double mutant, resulted in the additional distribution of Crumbs, an apical stalk membrane protein, to the lateral basal photoreceptor membrane. This phenotype was recapitulated by the knockdown of the catalytic subunit of Protein phosphatase 1, a known interactor with Bifocal. Taken together, these results expand our understanding of the molecular mechanisms underlying the generation of the two distinct photoreceptor apical domains.more » « less
-
Abstract Effective cellular signaling relies on precise spatial localization and dynamic interactions among proteins in specific subcellular compartments or niches, such as cell-to-cell contact sites and junctions. In plants, endogenous and pathogenic proteins gained the ability to target plasmodesmata, membrane-lined cytoplasmic connections, through evolution to regulate or exploit cellular signaling across cell wall boundaries. For example, the receptor-like membrane protein PLASMODESMATA-LOCATED PROTEIN 5 (PDLP5), a potent regulator of plasmodesmal permeability, generates feed-forward or feed-back signals important for plant immunity and root development. However, the molecular features that determine the plasmodesmal association of PDLP5 or other proteins remain largely unknown, and no protein motifs have been identified as plasmodesmal targeting signals. Here, we developed an approach combining custom-built machine-learning algorithms and targeted mutagenesis to examine PDLP5 in Arabidopsis thaliana and Nicotiana benthamiana. We report that PDLP5 and its closely related proteins carry unconventional targeting signals consisting of short stretches of amino acids. PDLP5 contains 2 divergent, tandemly arranged signals, either of which is sufficient for localization and biological function in regulating viral movement through plasmodesmata. Notably, plasmodesmal targeting signals exhibit little sequence conservation but are located similarly proximal to the membrane. These features appear to be a common theme in plasmodesmal targeting.more » « less
An official website of the United States government

