Chloroplast-localized translation for protein targeting in Chlamydomonas reinhardtii

Translation is localized within cells to target proteins to their proper locations. We asked whether translation occurs on the chloroplast surface in Chlamydomonas and, if so, whether it is involved in co-translational protein targeting, aligned spatially with localized translation by the bacterial-type ribosomes within this organelle, or both. Our results reveal a domain of the chloroplast envelope which is bound by translating ribosomes. Purified chloroplasts retained ribosomes and mRNAs encoding two chloroplast proteins specifically on this translation domain, but not a mRNA encoding a cytoplasmic protein. Ribosomes clusters were seen on this domain by electron tomography. Activity of the chloroplast-bound ribosomes is supported by results of the ribopuromycylation and puromycin-release assays. Co-translational chloroplast protein import is supported by nascent polypeptide dependency of the ribosome-chloroplast associations. This cytoplasmic translation domain aligns localized translation by organellar bacterial-type ribosomes in the chloroplast. This juxtaposition the dual translation systems facilitates the targeting and assembly of the polypeptide products.

Profiling subcellular localization of nuclear-encoded mitochondrial gene products in zebrafish

Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. The import of some, but not all, nuclear-encoded mitochondrial proteins begins with translation of messenger RNAs (mRNAs) on the surface of mitochondria. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a-/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to employ localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.

Cytosolic Quality Control of Mitochondrial Protein Precursors—The Early Stages of the Organelle Biogenesis

With few exceptions, proteins that constitute the proteome of mitochondria originate outside of this organelle in precursor forms. Such protein precursors follow dedicated transportation paths to reach specific parts of mitochondria, where they complete their maturation and perform their functions. Mitochondrial precursor targeting and import pathways are essential to maintain proper mitochondrial function and cell survival, thus are tightly controlled at each stage. Mechanisms that sustain protein homeostasis of the cytosol play a vital role in the quality control of proteins targeted to the organelle. Starting from their synthesis, precursors are constantly chaperoned and guided to reduce the risk of premature folding, erroneous interactions, or protein damage. The ubiquitin-proteasome system provides proteolytic control that is not restricted to defective proteins but also regulates the supply of precursors to the organelle. Recent discoveries provide evidence that stress caused by the mislocalization of mitochondrial proteins may contribute to disease development. Precursors are not only subject to regulation but also modulate cytosolic machinery. Here we provide an overview of the cellular pathways that are involved in precursor maintenance and guidance at the early cytosolic stages of mitochondrial biogenesis. Moreover, we follow the circumstances in which mitochondrial protein import deregulation disturbs the cellular balance, carefully looking for rescue paths that can restore proteostasis.

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.

Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

Quantitative Proteomics and Differential Protein Abundance Analysis after the Depletion of PEX3 from Human Cells Identifies Additional Aspects of Protein Targeting to the ER

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of around 10,000 different soluble and membrane proteins in humans. It involves the co- or post-translational targeting of precursor polypeptides to the ER, and their subsequent membrane insertion or translocation. So far, three pathways for the ER targeting of precursor polypeptides and four pathways for the ER targeting of mRNAs have been described. Typically, these pathways deliver their substrates to the Sec61 polypeptide-conducting channel in the ER membrane. Next, the precursor polypeptides are inserted into the ER membrane or translocated into the ER lumen, which may involve auxiliary translocation components, such as the TRAP and Sec62/Sec63 complexes, or auxiliary membrane protein insertases, such as EMC and the TMCO1 complex. Recently, the PEX19/PEX3-dependent pathway, which has a well-known function in targeting and inserting various peroxisomal membrane proteins into pre-existent peroxisomal membranes, was also found to act in the targeting and, putatively, insertion of monotopic hairpin proteins into the ER. These either remain in the ER as resident ER membrane proteins, or are pinched off from the ER as components of new lipid droplets. Therefore, the question arose as to whether this pathway may play a more general role in ER protein targeting, i.e., whether it represents a fourth pathway for the ER targeting of precursor polypeptides. Thus, we addressed the client spectrum of the PEX19/PEX3-dependent pathway in both PEX3-depleted HeLa cells and PEX3-deficient Zellweger patient fibroblasts by an established approach which involved the label-free quantitative mass spectrometry of the total proteome of depleted or deficient cells, as well as differential protein abundance analysis. The negatively affected proteins included twelve peroxisomal proteins and two hairpin proteins of the ER, thus confirming two previously identified classes of putative PEX19/PEX3 clients in human cells. Interestingly, fourteen collagen-related proteins with signal peptides or N-terminal transmembrane helices belonging to the secretory pathway were also negatively affected by PEX3 deficiency, which may suggest compromised collagen biogenesis as a hitherto-unknown contributor to organ failures in the respective Zellweger patients.

In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells

Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow mobilities. While in the cytoplasm the correlation times were in the 2–6 and 40–110 ms range for the fast and slow mobility populations respectively, in the cell nucleus they were 1–10 and 25–140 ms range, respectively. The fast mobility of DENV C in cytoplasmic and nuclear regions agreed with the diffusion coefficients from Brownian motion previously reported from correlation analysis. These studies provide the first evidence of DENV C shuttling from and to the nucleus in infected cells, opening new venues for antiviral interventions.

Monitoring Protein Import into the Endoplasmic Reticulum in Living Cells with Proximity Labeling

The proper trafficking of eukaryotic proteins is essential to cellular function. Genetic, environmental, and other stresses can induce protein mistargeting, and in turn threaten cellular protein homeostasis. Current methods for measuring protein mistargeting are difficult to translate to living cells, and thus the role of cellular signaling networks in stress-dependent protein mistargeting processes, such as ER pre-emptive quality control (ER pQC), are difficult to parse. Herein, we use genetically encoded peroxidases to characterize protein import into the endoplasmic reticulum (ER). We show that the ERHRP/cytAPEX pair provides good selectivity and sensitivity for identifying protein mistargeting, using the known ER pQC substrate transthyretin (TTR). Although ERHRP labeling induces formation of detergent-resistant TTR aggregates, this is minimized by using low ERHRP expression, without loss of labeling efficiency. cytAPEX labeling recovers TTR that is mistargeted as a consequence of Sec61 inhibition or ER stress-induced ER pQC. Furthermore, we demonstrate that stress-free activation of the ER stress-associated transcription factor ATF6 recapitulates the TTR import deficiency of ER pQC. Hence, proximity labeling is an effective strategy for characterizing factors that influence ER protein import in living cells.

The Spliced Leader RNA Silencing (SLS) Pathway in Trypanosoma brucei Is Induced by Perturbations of Endoplasmic Reticulum, Golgi Complex, or Mitochondrial Protein Factors: Functional Analysis of SLS-Inducing Kinase PK3

In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS.