Dissecting the interactions of PINK1 with the TOM complex in depolarized mitochondria

Mitochondria dysfunction is involved in the pathomechanism of many illnesses including Parkinson's disease. PINK1, which is mutated in some cases of familiar Parkinsonism, is a key component in the degradation of damaged mitochondria by mitophagy. The accumulation of PINK1 on the mitochondrial outer membrane (MOM) of compromised organelles is crucial for the induction of mitophagy, but the molecular mechanism of this process is still unresolved. Here, we investigate the association of PINK1 with the TOM complex. We demonstrate that PINK1 heavily relies on the import receptor TOM70 for its association with mitochondria and directly interacts with this receptor. The structural protein TOM7 appears to play only a moderate role in PINK1 association with the TOM complex, probably due to its role in stabilizing this complex. PINK1 requires the TOM40 pore lumen for its stable interaction with the TOM complex and apparently remains there during its further association with the MOM. Overall, this study provides new insights on the role of the individual TOM subunits in the association of PINK1 with the MOM of depolarized mitochondria.

A unified model for the biogenesis of mitochondrial outer membrane multi-span proteins

The mitochondrial outer membrane (MOM) harbors proteins that traverse the membrane via several helical segments, so-called multi-span proteins. Two contradicting mechanisms were suggested to describe their integration into the MOM. The first proposes that the mitochondrial import (MIM) complex facilitates this process and functions as an insertase, whereas the second suggests that such proteins can integrate into the lipid phase without the assistance of import factors in a process that is enhanced by phosphatidic acid. To resolve this discrepancy and obtain new insights on the biogenesis of these proteins, we addressed this issue using yeast mitochondria and the multi-span protein Om14. Testing different truncation variants, we show that only the full-length protein contains all the required information that assure targeting specificity. Employing a specific insertion assay and several single and double deletion strains, we show that neither the import receptor Tom70 nor any other protein with a cytosolically exposed domain have a crucial contribution to the biogenesis process. We further demonstrate that Mim1 and Porin are required for optimal membrane integration of Om14 but none of them is absolutely required. Unfolding of the newly synthesized protein, its optimal hydrophobicity, as well as higher fluidity of the membrane dramatically enhanced the import capacity of Om14. Collectively, our findings suggest that MOM multi-span proteins can follow different biogenesis pathways in which proteinaceous elements and membrane behavior contribute to a variable extent to the combined efficiency.

Mistargeting of hydrophobic mitochondrial proteins activates a nucleus-mediated posttranscriptional quality control pathway in trypanosomes

Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all mitochondria.

Global kinome profiling reveals DYRK1A as critical activator of the human mitochondrial import machinery

The translocase of the outer mitochondrial membrane TOM constitutes the organellar entry gate for nearly all precursor proteins synthesized on cytosolic ribosomes. Thus, TOM presents the ideal target to adjust the mitochondrial proteome upon changing cellular demands. Here, we identify that the import receptor TOM70 is targeted by the kinase DYRK1A and that this modification plays a critical role in the activation of the carrier import pathway. Phosphorylation of TOM70Ser91 by DYRK1A stimulates interaction of TOM70 with the core TOM translocase. This enables transfer of receptor-bound precursors to the translocation pore and initiates their import. Consequently, loss of TOM70Ser91 phosphorylation results in a strong decrease in import capacity of metabolite carriers. Inhibition of DYRK1A impairs mitochondrial structure and function and elicits a protective transcriptional response to maintain a functional import machinery. The DYRK1A-TOM70 axis will enable insights into disease mechanisms caused by dysfunctional DYRK1A, including autism spectrum disorder, microcephaly and Down syndrome.

Nuclear Import Receptors and hnRNP K Mediates Nuclear and Stress Granule Localization of SIRLOIN

The majority of lncRNAs and a small fraction of mRNAs localize in the cell nucleus to exert their functions. A SIRLOIN RNA motif was previously reported to drive its nuclear localization by the RNA-binding protein hnRNP K. However, the underlying mechanism remains unclear. Here, we report crystal structures of hnRNP K in complex with SIRLOIN, and with the nuclear import receptor (NIR) Impα1, respectively. The protein hnRNP K bound to SIRLOIN with multiple weak interactions, and interacted Impα1 using an independent high-affinity site. Forming a complex with hnRNP K and Impα1 was essential for the nuclear and stress granule localization of SIRLOIN in semi-permeabilized cells. Nuclear import of SIRLOIN enhanced with increasing NIR concentrations, but its stress granule localization peaked at a low NIR concentration. Collectively, we propose a mechanism of SIRLOIN localization, in which NIRs functioned as drivers/regulators, and hnRNP K as an adaptor.

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics.

The Nuclear Transport Protein Importin-5: A Promising Target in Oncology and Virology

Cellular homeostasis importantly relies on the correct nucleoplasmic distribution of a large number of RNA molecules and proteins, which are shuttled by specialized transport receptors. The nuclear import receptor importin-5, also called IPO5, RanBP5 or karyopherin β3, mediates the translocation of proteins to the nucleus, and thus regulates critical signaling pathways and cellular functions. The normal function of IPO5 appears to be disrupted in cancer cells due to aberrant overexpression. IPO5 also demonstrated a pivotal role in viral replication. The constant increasing number of publications shows an interest within the scientific community as a therapeutic target due to its pivotal role in protein trafficking.

Transportin-1: A Nuclear Import Receptor with Moonlighting Functions

Transportin-1 (Trn1), also known as karyopherin-β2 (Kapβ2), is probably the best-characterized nuclear import receptor of the karyopherin-β family after Importin-β, but certain aspects of its functions in cells are still puzzling or are just recently emerging. Since the initial identification of Trn1 as the nuclear import receptor of hnRNP A1 ∼25 years ago, several molecular and structural studies have unveiled and refined our understanding of Trn1-mediated nuclear import. In particular, the understanding at a molecular level of the NLS recognition by Trn1 made a decisive step forward with the identification of a new class of NLSs called PY-NLSs, which constitute the best-characterized substrates of Trn1. Besides PY-NLSs, many Trn1 cargoes harbour NLSs that do not resemble the archetypical PY-NLS, which complicates the global understanding of cargo recognition by Trn1. Although PY-NLS recognition is well established and supported by several structures, the recognition of non-PY-NLSs by Trn1 is far less understood, but recent reports have started to shed light on the recognition of this type of NLSs. Aside from its principal and long-established activity as a nuclear import receptor, Trn1 was shown more recently to moonlight outside nuclear import. Trn1 has for instance been caught in participating in virus uncoating, ciliary transport and in modulating the phase separation properties of aggregation-prone proteins. Here, we focus on the structural and functional aspects of Trn1-mediated nuclear import, as well as on the moonlighting activities of Trn1.

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81–120, 190–210, and 380–389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21–50 and 380–389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380–389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.