Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, ...others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state.
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► MITRAC is a cytochrome c oxidase assembly intermediate ► MITRAC regulates mitochondrial translation in human mitochondria ► TIM21 is a constituent of MITRAC and presequence translocase complexes ► TIM21 ushers imported subunits of complexes I and IV to assembly intermediates
The mitochondrial-inner-membrane translocase subunit TIM21 ushers cytoplasmically translated components of electron transport chain complexes (ETCs) away from the translocase and promotes their integration into ETC assembly intermediates that also contain mitochondrially translated subunits.
While cilia are recognized as important signaling organelles, the extent of ciliary functions remains unknown because of difficulties in cataloguing proteins from mammalian primary cilia. We present ...a method that readily captures rapid snapshots of the ciliary proteome by selectively biotinylating ciliary proteins using a cilia-targeted proximity labeling enzyme (cilia-APEX). Besides identifying known ciliary proteins, cilia-APEX uncovered several ciliary signaling molecules. The kinases PKA, AMPK, and LKB1 were validated as bona fide ciliary proteins and PKA was found to regulate Hedgehog signaling in primary cilia. Furthermore, proteomics profiling of Ift27/Bbs19 mutant cilia correctly detected BBSome accumulation inside Ift27−/− cilia and revealed that β-arrestin 2 and the viral receptor CAR are candidate cargoes of the BBSome. This work demonstrates that proximity labeling can be applied to proteomics of non-membrane-enclosed organelles and suggests that proteomics profiling of cilia will enable a rapid and powerful characterization of ciliopathies.
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•APEX labeling enables proteomic analyses of a non-membrane-enclosed compartment•Cilia-APEX identifies the kinases PKA, AMPK, and LKB1 in primary cilia•PKA functions inside cilia to phosphorylate GLI3 and regulate Hedgehog signaling•Proteomic profiling of Ift27/Bbs19 cilia detects ciliary accumulation of BBSome
Primary cilia organize cellular signaling events in a specialized microenvironment. Mick et al. apply proximity labeling using cilia-APEX to study the ciliary proteome. They uncover unexpected signaling molecules, including kinases PKA and AMPK, inside cilia and further use a proteomic profiling approach to unravel molecular defects of Ift27/Bbs19 mutant cilia.
The primary cilium projects from the surface of most vertebrate cells, where it senses extracellular signals to regulate diverse cellular processes during tissue development and homeostasis. ...Dysfunction of primary cilia underlies the pathogenesis of severe diseases, commonly referred to as ciliopathies. Primary cilia contain a unique protein repertoire that is distinct from the cell body and the plasma membrane, enabling the spatially controlled transduction of extracellular cues. G-protein coupled receptors (GPCRs) are key in sensing environmental stimuli that are transmitted via second messenger signaling into a cellular response. Here, we will give an overview of the role of GPCR signaling in primary cilia, and how ciliary GPCR signaling can be targeted by pharmacology, chemogenetics, and optogenetics.
Mitochondria maintain genome and translation machinery to synthesize a small subset of subunits of the oxidative phosphorylation system. To build up functional enzymes, these organellar gene products ...must assemble with imported subunits that are encoded in the nucleus. New findings on the early steps of cytochrome c oxidase assembly reveal how the mitochondrial translation of its core component, cytochrome c oxidase subunit 1 (Cox1), is directly coupled to the assembly of this respiratory complex.
Proximity labeling by ascorbate peroxidase (APEX) requires appropriate experimental setups that generate sufficient signal over background as a prerequisite for downstream analyses by mass ...spectrometry. Cell culture-based systems are easily accessible, yet, for proximity labeling of small structures must be carefully optimized in order to give satisfying results. How to establish and characterize APEX cell lines will be the topic of this chapter.
Compartmentalization of cellular signaling forms the molecular basis of cellular behavior. The primary cilium constitutes a subcellular compartment that orchestrates signal transduction independent ...from the cell body. Ciliary dysfunction causes severe diseases, termed ciliopathies. Analyzing ciliary signaling has been challenging due to the lack of tools to investigate ciliary signaling. Here, we describe a nanobody-based targeting approach for optogenetic tools in mammalian cells and in vivo in zebrafish to specifically analyze ciliary signaling and function. Thereby, we overcome the loss of protein function observed after fusion to ciliary targeting sequences. We functionally localized modifiers of cAMP signaling, the photo-activated adenylyl cyclase bPAC and the light-activated phosphodiesterase LAPD, and the cAMP biosensor mlCNBD-FRET to the cilium. Using this approach, we studied the contribution of spatial cAMP signaling in controlling cilia length. Combining optogenetics with nanobody-based targeting will pave the way to the molecular understanding of ciliary function in health and disease.
Regulation of eukaryotic cytochrome oxidase assembly occurs at the level of Cox1 translation, its central mitochondria-encoded subunit. Translation of COX1 messenger RNA is coupled to complex ...assembly in a negative feedback loop: the translational activator Mss51 is thought to be sequestered to assembly intermediates, rendering it incompetent to promote translation. In this study, we identify Coa3 (cytochrome oxidase assembly factor 3; Yjl062w-A), a novel regulator of mitochondrial COX1 translation and cytochrome oxidase assembly. We show that Coa3 and Cox14 form assembly intermediates with newly synthesized Cox1 and are required for Mss51 association with these complexes. Mss51 exists in equilibrium between a latent, translational resting, and a committed, translation-effective, state that are represented as distinct complexes. Coa3 and Cox14 promote formation of the latent state and thus down-regulate COX1 expression. Consequently, lack of Coa3 or Cox14 function traps Mss51 in the committed state and promotes Cox1 synthesis. Our data indicate that Coa1 binding to sequestered Mss51 in complex with Cox14, Coa3, and Cox1 is essential for full inactivation.
The primary cilium is a signaling compartment that interprets Hedgehog signals through changes of its protein, lipid, and second messenger compositions. Here, we combine proximity labeling of cilia ...with quantitative mass spectrometry to unbiasedly profile the time-dependent alterations of the ciliary proteome in response to Hedgehog. This approach correctly identifies the three factors known to undergo Hedgehog-regulated ciliary redistribution and reveals two such additional proteins. First, we find that a regulatory subunit of the cAMP-dependent protein kinase (PKA) rapidly exits cilia together with the G protein-coupled receptor GPR161 in response to Hedgehog, and we propose that the GPR161/PKA module senses and amplifies cAMP signals to modulate ciliary PKA activity. Second, we identify the phosphatase Paladin as a cell type-specific regulator of Hedgehog signaling that enters primary cilia upon pathway activation. The broad applicability of quantitative ciliary proteome profiling promises a rapid characterization of ciliopathies and their underlying signaling malfunctions.
Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. ...Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome—the trafficking complex in charge of retrieving GPCRs from cilia—engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.
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•Several readers of K63-linked ubiquitin chains accumulate in Bbs−/− mammalian cilia•The K63-linked ubiquitin chains reader Target of Myb1-like 2 (TOM1L2) directly binds to the BBSome•Targeted disruption of the BBSome-TOM1L2 interaction blocks G protein-coupled receptor exits from cilia•Chlamydomonas TOM1 is required for the removal of ubiquitinated proteins from cilia
Shinde et al. find that the BBSome does not directly recognize lysine 63-linked ubiquitin chains. Instead, proteomics profiling, focused screens, biochemical mapping, and targeted disruption identify the ancestral ESCRT-0 protein Target of Myb1-like 2 (TOM1L2) as the adaptor that enables the BBSome to select ubiquitinated G protein-coupled receptors for removal from cilia.
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