Protein Sorting Pathway Research Articles

Transcriptomic landscapes reveal development-related physiological processes in the two-spotted spider mite, Tetranychus urticae.

The two-spotted spider mite (Tetranychus urticae Koch, TSSM) is recognized as one of the most problematic spider mite pests. However, the precise gene expression patterns across its key developmental stages remain elusive. Here, we performed a comprehensive transcriptome analysis of TSSM eggs, nymphs and adult females using publicly available RNA sequencing (RNA-seq) data to elucidate the overarching transcriptomic differences between these developmental stages. Principal component analysis and hierarchical clustering analysis unveiled distinct separations among samples across different developmental stages, regardless of their Wolbachia infection status. Differential expression analysis revealed 4,089,2,762, and 1,282 core genes specifically enriched in eggs, nymphs, and adults, respectively. KEGG and GO enrichment analyses showed upregulation of genes in eggs are associated with proteolysis, Wnt signaling pathway, DNA transcription, RNA biosynthetic and metabolic processes, as well as protein folding, sorting, and degradation pathways. Meanwhile, nymphs exhibited increased abundance of genes related to chitin/amino sugar metabolic processes, G protein-coupled receptor signaling pathways, monoatomic ion transport, and neurotransmitter transport pathways. Pathways involving sphingolipid and carbohydrate metabolic processes, proteolysis, lipid transport, and localization were particularly enriched in older females. Altogether, our findings suggest that the egg stage exhibits higher activity in cell differentiation processes, the nymph stage is more involved in chitin development, and the adult stage shows increased metabolic and reproductive activity. This study enhances our understanding of the molecular mechanisms underlying TSSM development and paves the way for further research into the intricate physiological processes of TSSM.

Cryo-EMstructure of SRP68/72 reveals an extended dimerization domain with RNA-binding activity.

The signal recognition particle (SRP) is a critical component in protein sorting pathways in all domains of life. Human SRP contains six proteins bound to the 7S RNA and their structures and functions have been mostly elucidated. The SRP68/72 dimer is the largest SRP component and is essential for SRP function. Although the structures of the SRP68/72 RNA binding and dimerization domains have been previously reported, the structure and function of large portions of the SRP68/72 dimer remain unknown. Here, we analyse full-length SRP68/72 using cryo-EM and report that SRP68/72 depend on each other for stability and form an extended dimerization domain. This newly observed dimerization domain is both a protein- and RNA-binding domain. Comparative analysis with current structural models suggests that this dimerization domain undergoes dramatic translocation upon SRP docking onto SRP receptor and eventually comes close to the Alu domain. We propose that the SRP68/72 dimerization domain functions by binding and detaching the Alu domain and SRP9/14 from the ribosomal surface, thus releasing elongation arrest upon docking onto the ER membrane.

A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting.

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.

New insights into the physiological effects of structural environmental enrichment on black sea bream (Acanthopagrus schlegelii) based on a combined analysis of transcriptomics and metabolomics

Environmental enrichment (EE) has been generally accepted as an effective and promising way to enhance the performance and improve the welfare of animals kept in captivity. Evidence for the effects of EE on fish has accumulated rapidly, but little is known about its underlying mechanisms. In this study, black sea bream (Acanthopagrus schlegelii) were reared under enriched and enrichment-free conditions for 30 (post-larval) and 60 (juvenile) days. Physiological, transcriptome, and metabolome data were compared to investigate potential rearing improvements, physiological responses to acute stress, and the underlying mechanisms. The results showed that fish reared under enriched conditions had significantly better growth performance, a higher survival rate, and lower basal cortisol levels. Meanwhile, the cortisol, lactic acid, malondialdehyde (MDA), and total antioxidant capacity (T-AOC) levels in fish subjected to acute stress suggested that EE-reared individuals presented greater stress resistance. However, the effect of EE may decrease with the extension of treatment time for larger fish. The transcriptome analysis showed that energy-related pathways (including oxidative phosphorylation, the citrate cycle, and glycolysis/gluconeogenesis) as well as protein folding, sorting, and degradation-related pathways (protein processing in the endoplasmic reticulum and proteasome pathways); replication and repair related pathways (including pathways involved in nucleotide excision repair, base excision repair, mismatch repair, and homologous recombination); and immune-related pathways (complement and coagulation cascades pathway and the IL-17 signaling pathway) were significantly altered in the EE-reared individuals. In addition, integrated analysis of metabolome and transcriptome data revealed marked enrichment of transcription-associated metabolites involved in cortisol synthesis and secretion, taste transduction, olfactory transduction, and protein digestion and absorption pathways. Our results suggested that EE may influence the growth, fitness, and stress resistance of A. schlegelii by regulating a series of biological processes including energy metabolism, cortisol synthesis, antioxidant enzyme activities, protein folding, DNA replication and repair, and complement and coagulation cascades.

A DUF3844 domain-containing protein is required for vacuolar protein sorting in Schizosaccharomyces pombe.

Protein trafficking to vacuoles in plants and fungi, and to lysosomes in animals, is essential for the maintenance of cellular homeostasis. In Saccharomyces cerevisiae, the vacuolar protein sorting (VPS) pathway has been well studied by using vacuolar carboxypeptidase Y (CPY) as a model, and many VPS genes have been identified. By contrast, the vacuolar protein trafficking pathway in Schizosaccharomyces pombe remains poorly understood. In this study, we identified a novel VPS gene (SPBC1709.03) in S. pombe that is broadly conserved in fungi, but not in S. cerevisiae. Owing to its DUF3844 domain of unknown function, the gene was named vps3844. Disruption mutants of vps3844 had defects in both CPY sorting and incorporation of FM4-64 dye into the vacuolar membrane. Partial deletion analysis of the Vps3844 protein revealed that, within the DUF3844 domain, the region comprising amino acids 354 to 380 is important for protein trafficking to the vacuole. Our findings represent the first report of a VPS gene involved in vacuolar transport that is conserved in fungi, particularly S. pombe, but lacks representation in S. cerevisiae.

Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence.

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson's Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

The identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in theVPS35gene has been reported to be linked to type 17 Parkinson’s Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Comparative proteome and volatile metabolome analysis of Aspergillus oryzae 3.042 and Aspergillus sojae 3.495 during koji fermentation

Aspergillus oryzae 3.042 and Aspergillus sojae 3.495 are crucial starters for fermented soybean foods since their abundant secreted enzymes. This study aimed to compare the differences in protein secretion between A. oryzae 3.042 and A. sojae 3.495 during the soy sauce koji fermentation and the effect on volatile metabolites to understand the fermentation characteristics of the strains better. Label-free proteomics detected 210 differentially expressed proteins (DEPs) enriched in amino acid metabolism and protein folding, sorting and degradation pathways. Subsequently, extracellular enzyme analysis showed that three peptidases, including peptide hydrolase, dipeptidyl aminopeptidase, and peptidase S41, were up-regulated in A. sojae 3.495. Seven carbohydrases, including α-galactosidase, endo-arabinase, β-glucosidase, α-galactosidase, α-glucuronidase, arabinan-endo 1,5-α-l-arabinase, and endo-1,4-β-xylanase were up-regulated in A. oryzae 3.042, contributing to the difference in enzyme activity. Significantly different extracellular enzymes influenced the content of volatile alcohols, aldehydes and esters such as (R, R)-2,3-butanediol, 1-hexanol, hexanal, decanal, ethyl l-lactate and methyl myristate in both strains, which affected the type of aroma of koji. Overall, this study revealed the differences in molecular mechanisms between A. oryzae 3.042 and A. sojae 3.495 under solid-state fermentation, providing a reference for targeted enhancement strains.

Gene Expression Analysis of Adapted Insect Cells during Influenza VLP Production Using RNA-Sequencing.

Adaptive laboratory evolution has been used to improve production of influenza hemagglutinin (HA)-displaying virus-like particles (VLPs) in insect cells. However, little is known about the underlying biological mechanisms promoting higher HA-VLP expression in such adapted cell lines. In this article, we present a study of gene expression patterns associated with high-producer insect High Five cells adapted to neutral pH, in comparison to non-adapted cells, during expression of influenza HA-VLPs. RNA-seq shows a decrease in the amount of reads mapping to host cell genomes along infection, and an increase in those mapping to baculovirus and transgenes. A total of 1742 host cell genes were found differentially expressed between adapted and non-adapted cells throughout infection, 474 of those being either up- or down-regulated at both time points evaluated (12 and 24 h post-infection). Interestingly, while host cell genes were found up- and down-regulated in an approximately 1:1 ratio, all differentially expressed baculovirus genes were found to be down-regulated in infected adapted cells. Pathway analysis of differentially expressed genes revealed enrichment of ribosome biosynthesis and carbohydrate, amino acid, and lipid metabolism. In addition, oxidative phosphorylation and protein folding, sorting and degradation pathways were also found to be overrepresented. These findings contribute to our knowledge of biological mechanisms of insect cells during baculovirus-mediated transient expression and will assist the identification of potential engineering targets to increase recombinant protein production in the future.

A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways.

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

VPS34 complexes from a structural perspective

VPS34 phosphorylates phosphatidylinositol to produce PtdIns3P and is the progenitor of the phosphoinositide 3-kinase (PI3K) family. VPS34 has a simpler domain organization than class I PI3Ks, which belies the complexity of its quaternary organization, with the enzyme always functioning within larger assemblies. PtdIns3P recruits specific recognition modules that are common in protein-sorting pathways, such as autophagy and endocytic sorting. It is best characterized in two heterotetramers, complexes I and II. Complex I is composed of VPS34, VPS15, Beclin 1, and autophagy-related gene (ATG)14L, whereas complex II replaces ATG14L with UVRAG. Because VPS34 can form a component of several distinct complexes, it enables independent regulation of various pathways that are controlled by PtdIns3P. Complexes I and II are critical for early events in autophagy and endocytic sorting, respectively. Autophagy has a complex association with cancer. In early stages, it inhibits tumorigenesis, but in later stages, it acts as a survival factor for tumors. Recently, various disease-associated somatic mutations were found in genes encoding complex I and II subunits. Lipid kinase activities of the complexes are also influenced by posttranslational modifications (PTMs). Mapping PTMs and somatic mutations on three-dimensional models of the complexes suggests mechanisms for how these affect VPS34 activity.

The intrinsically disordered nature of the peroxisomal protein translocation machinery.

Despite having a membrane that is impermeable to all but the smallest of metabolites, peroxisomes acquire their newly synthesized (cytosolic) matrix proteins in an already folded conformation. In some cases, even oligomeric proteins have been reported to translocate the organelle membrane. The protein sorting machinery that accomplishes this feat must be rather flexible and, unsurprisingly, several of its key components have large intrinsically disordered domains. Here, we provide an overview on these domains and their interactions trying to infer their functional roles in this protein sorting pathway.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta.

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Discovery of Several Novel Targets that Enhance β-Carotene Production in Saccharomyces cerevisiae.

β-Carotene is the precursor of vitamin A, and also exhibits multiple pharmaceutical functions by itself. In comparison to chemical synthesis, the production of β-carotene in microbes by metabolic engineering strategy is relatively inexpensive. Identifying genes enhancing β-carotene production in microbes is important for engineering a strain of producing higher yields of β-carotene. Most of previous efforts in identifying the gene targets have focused on the isoprenoid pathway where the β-carotene biosynthesis belongs. However, due to the complex interactions between metabolic fluxes, seemingly irrelevant genes that are outside the isoprenoid pathway might also affect β-carotene biosynthesis. To this end, here we provided an example that several novel gene targets, which are outside the isoprenoid pathway, have improving effects on β-carotene synthesis in yeast cells, when they were over-expressed. Among these targets, the class E protein of the vacuolar protein-sorting pathway (Did2) led to the highest improvement in β-carotene yields, which was 2.1-fold to that of the corresponding control. This improvement was further explained by the observation that the overexpression of the DID2 gene generally boosted the transcriptions of β-carotene pathway genes. The mechanism by which the other targets improve the production of β-carotene is discussed.

Investigation of a Mitochondrial Twin Arginine Transport Pathway in Arabidopsis thaliana

Most proteins are synthesized at a location different to their site of function. For example, most of the proteins found in cell organelles such as mitochondria or chloroplasts are encoded by nuclear genes, which are transcribed in the nucleus and translated in the cytoplasm. Cells therefore, must have efficient protein transport processes to allow proteins to cross membranes such as those bounding cell organelles like mitochondria or chloroplasts. Proteins destined for organelles are synthesized as larger molecular weight precursors, containing targeting sequences, which direct the precursor to the appropriate transport apparatus at the membrane.One such vital protein transport pathway is the Tat (Twin Arginine Translocation) pathway. The Tat pathway has been found in a wide variety of organisms including bacteria, archaea and eukaryotic organelles such as the chloroplast. The chloroplast Tat pathway (or analogous Tat pathways in bacteria) is capable of transporting fully folded proteins across membranes using only a proton motive force instead of ATP for energy. It is composed of three protein components, namely cpTatC (TatC), Tha4 (TatA) and Hcf106 (TatB) that work together to allow the transport of precursor proteins containing an obligate twin arginine motif in the targeting sequence.However, recent experiments using Nicotiana tabacum cells showed an up‐regulation of a mitochondrial gene coding for a mtTatC protein when the cells was subjected to salicylic acid treatment, which mimics a defense response in the plant to attack by pests. However, single nuclear genes encode both of the other Tat components. Analogous transport pathways in certain Gram (+) bacteria demonstrate that a minimal functional Tat pathway is composed of TatC and TatA (Tha4 homologue). We have confirmed a similar upregulation under stress of salicylic acid of the putative mitochondrial tatC gene in Arabidopsis thaliana. The presence and upregulation under stress of the mttatC gene suggests the possible presence of a mitochondrial Tat pathway, which would require the dual localization of a TatA homology, such as Tha4.To test the possible dual localization of Tha4 to mitochondrial, we have transformed Arabidopsis thaliana with Tha4‐GFP constructs and looked for its localization in the presence or absence of various biotic or abiotic stresses. In addition, we will look at changes in gene expression using RT‐PCR technique to look for Tha4 upregulation in the presence or absence of the stress.Evaluating the presence of a mitochondrial Tat pathway will shed light on a unique protein sorting and transport mechanism in plant mitochondria, a facility previously thought to be lost during the mitochondrial evolutionary process. Therefore investigating this protein sorting pathway in mitochondria may further our understanding of acquired innate immunity within flowering plant species.

Identification of a Functional Plasmodesmal Localization Signal in a Plant Viral Cell-To-Cell-Movement Protein.

ABSTRACTOur fundamental knowledge of the protein-sorting pathways required for plant cell-to-cell trafficking and communication via the intercellular connections termed plasmodesmata has been severely limited by the paucity of plasmodesmal targeting sequences that have been identified to date. To address this limitation, we have identified the plasmodesmal localization signal (PLS) in the Tobacco mosaic virus (TMV) cell-to-cell-movement protein (MP), which has emerged as the paradigm for dissecting the molecular details of cell-to-cell transport through plasmodesmata. We report here the identification of a bona fide functional TMV MP PLS, which encompasses amino acid residues between positions 1 and 50, with residues Val-4 and Phe-14 potentially representing critical sites for PLS function that most likely affect protein conformation or protein interactions. We then demonstrated that this PLS is both necessary and sufficient for protein targeting to plasmodesmata. Importantly, as TMV MP traffics to plasmodesmata by a mechanism that is distinct from those of the three plant cell proteins in which PLSs have been reported, our findings provide important new insights to expand our understanding of protein-sorting pathways to plasmodesmata.

Structural and Functional Insights into Small, Glutamine-Rich, Tetratricopeptide Repeat Protein Alpha.

The small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA) is an emerging player in the quality control of secretory and membrane proteins mislocalized to the cytosol, with established roles in tail-anchored (TA) membrane protein biogenesis. SGTA consists of three structural domains with individual functions, an N-terminal dimerization domain that assists protein sorting pathways, a central tetratricopeptide repeat (TPR) domain that mediates interactions with heat-shock proteins, proteasomal, and hormonal receptors, and viral proteins, and a C-terminal glutamine rich region that binds hydrophobic substrates. SGTA has been linked to viral lifecycles and hormone receptor signaling, with implications in the pathogenesis of various disease states. Thus far, a range of biophysical techniques have been employed to characterize SGTA structure in some detail, and to investigate its interactions with binding partners in different biological contexts. A complete description of SGTA structure, together with further investigation into its function as a co-chaperone involved quality control, could provide us with useful insights into its role in maintaining cellular proteostasis, and broaden our understanding of mechanisms underlying associated pathologies. This review describes how some structural features of SGTA have been elucidated, and what this has uncovered about its cellular functions. A brief background on the structure and function of SGTA is given, highlighting its importance to biomedicine and related fields. The current level of knowledge and what remains to be understood about the structure and function of SGTA is summarized, discussing the potential direction of future research.

Sortilin-related receptor CNS expressed 2 (SorCS2) is localized to Bunina bodies in amyotrophic lateral sclerosis

Sortilin-related receptor CNS expressed 2 (SorCS2) is one of the vacuolar protein sorting 10 family proteins (VPS10Ps) that have pleiotropic roles in protein trafficking and intracellular and intercellular signaling. Bunina bodies (BBs) are specifically detected in the lower motor neurons in patients with amyotrophic lateral sclerosis (ALS). BBs are immunolabeled with antibodies against cystatin C, transferrin and peripherin and are considered to originate from the endoplasmic reticulum, which is part of the protein sorting pathway. The present study investigated whether VPS10Ps are involved in the formation of BBs in ALS. We immunohistochemically examined the spinal cord from patients with ALS and control subjects using antibodies against VPS10Ps (sortilin, SorLA, SorCS1, SorCS2 and SorCS3). In normal controls, antibodies against VPS10Ps immunolabeled the cytoplasm of anterior horn cells in a fine granular pattern. In ALS, almost all BBs (95.1%) were strongly immunopositive for SorCS2, and immunoreativity for sortilin and SorLA was decreased in anterior horn cells. These findings suggest that VPS10Ps may be involved in the disease process of ALS.

Insight into the biogenesis of tax nuclear speckles using comparative interactome analysis

HTLV-1 Tax protein displays pleotropic activities that regulate many aspects of virus biology, host-pathogen interaction and disease development. Tax performs these various functions via protein-protein interactions and sub cellular compartmentalization appears to regulate which activities are engaged. We previously identified a novel nuclear body, Tax Speckled Structures (TSS), the biogenesis of which supports the sequestration of cellular DNA Damage Response factors by Tax to chromatin. To identify cellular proteins that drive localization of Tax to chromatin “speckles”, we conducted a Comparative Interactome Analysis of Tax and a Tax mutant missing the TSS localization signal (Tax-TSLS). Tandem affinity tagged Tax and mutant proteins were expressed in eukaryotic cells. Cytoplasmic, nuclear and chromatin fractions were generated and protein interactomes isolated by affinity purification. Those proteins that preferentially bound full-length Tax over Tax-TSLS were defined as the TSLS-specific interactome. Quantitative Ultra-high resolution tandem mass spectrometry was employed to establish the most comprehensive map of Tax-interacting proteins to date. We employed bioinformatics approaches to compare Tax interactomes and identify the functional pathways and interaction networks unique to proteins that bound the TSLS-defined region. The top functional networks for proteins that bound to the TSLS were involved in chromosomal alignment, congression, segregation, remodeling and modification. The overwhelming majority were known components of four chromatin functional super-complexes condensin I, kinesin, SWI/SNF and NURF. We also examined the protein interactions that were shared by full length and mutant Tax. These included significant interaction with DNA damage, cell cycle check point, NFkB signaling, and protein sorting pathways. A comparative analysis between the sub cellular fractions revealed that DNA damage and checkpoint signaling interactions were enriched in the nucleus/chromatin. These results demonstrate that Tax initiates TSS through binding of chromatin complexes and bridges the recruitment of DNA damage response and checkpoint signaling proteins.

Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol.

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.