Project description:Nanoflow liquid chromatography-mass spectrometry is key to enabling in-depth proteome profiling of trace samples such as single cells, but these separations can lack robustness due to the use of narrow-bore columns that are susceptible to clogging. In the case of single-cell proteomics, offline cleanup steps are generally omitted to avoid losses to additional surfaces, and online solid-phase extraction/trap columns frequently provide the only opportunity to remove salts and insoluble debris before the sample is introduced to the analytical column. Trap columns are traditionally short, packed columns used to load and concentrate analytes at flow rates greater than those employed in analytical columns, and since these first encounter the uncleaned sample mixture, trap columns are also susceptible to clogging. We hypothesized that clogging could be avoided by using large-bore porous layer open tubular trap columns (PLOTrap). The low back pressure ensured that the PLOTraps could also serve as the sample loop, thus allowing sample cleanup and injection with a single 6-port valve. We found that PLOTraps could effectively remove debris to avoid column clogging. We also evaluated multiple stationary phases and PLOTrap diameters to optimize performance in terms of peak widths and sample loading capacities. Optimized PLOTraps were compared to conventional packed trap columns operated in forward and backflush modes, and were found to have similar chromatographic performance of backflushed traps while providing improved debris removal for robust analyses of trace samples.
Project description:Mitochondrial biogenesis requires the import of many nuclear-encoded proteins across the Translocase of Outer Membrane (TOM), and the Translocase of Inner Membrane (TIM) 22 or 23 complexes. Protein import defects cannot only impair mitochondrial function but also cause mitochondrial Precursor Overaccumulation Stress (mPOS) in the cytosol. Recent studies showed that specific mutations in the nuclear-encoded Adenine Nucleotide Translocase 1 (ANT1) cause musculoskeletal and neurological diseases by clogging TOM and TIM22 and inducing mPOS. Here, we found that overexpression of MFB1, encoding the mitochondrial F-box protein 1, suppresses cell growth defect caused by a clogger allele of AAC2, the yeast homolog of Ant1. Disruption of MFB1 synergizes with a clogger allele of aac2 to inhibit cell growth. This is accompanied by increased retention of mitochondrial proteins in the cytosol, suggesting a severe defect in mitochondrial protein import. Proximity-dependent biotin identification (BioID) suggested that Mfb1 may interact with Tom22, a component of the TOM complex. Loss of MFB1 under clogging conditions activates genes encoding cytosolic chaperones including Hsp31. Interestingly, disruption of HSP31 creates a synthetic lethality with protein import clogging under respiring conditions. We propose that Mfb1 functions to maintain mitochondrial protein import competency under clogging conditions, whereas Hsp31 plays an important role in protecting the cytosol against mPOS. Mutations in human mitochondria-associated F-box proteins (e.g., Fbxo7) and the human Hsp31 homolog, DJ-1, are known to cause early-onset Parkinson’s disease. Our work may improve the understanding of how these mutations cause neurodegeneration.
Project description:Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in adenine nucleotide translocase 1 (Ant1), and its yeast homolog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of Ant1’s nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/Ant1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger Ant1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy Ant1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.
Project description:Mitochondrial biogenesis requires the import of many nuclear-encoded proteins across the Translocase of Outer Membrane (TOM), and the Translocase of Inner Membrane (TIM) 22 or 23 complexes. Protein import defects cannot only impair mitochondrial function but also cause mitochondrial Precursor Overaccumulation Stress (mPOS) in the cytosol. Recent studies showed that specific mutations in the nuclear-encoded Adenine Nucleotide Translocase 1 (ANT1) cause musculoskeletal and neurological diseases by clogging TOM and TIM22 and inducing mPOS. Here, we found that overexpression of MFB1, encoding the mitochondrial F-box protein 1, suppresses cell growth defect caused by a clogger allele of AAC2, the yeast homolog of Ant1. Disruption of MFB1 synergizes with a clogger allele of aac2 to inhibit cell growth. This is accompanied by increased retention of mitochondrial proteins in the cytosol, suggesting a severe defect in mitochondrial protein import. Proximity-dependent biotin identification (BioID) suggested that Mfb1 may interact with Tom22, a component of the TOM complex. Loss of MFB1 under clogging conditions activates genes encoding cytosolic chaperones including Hsp31. Interestingly, disruption of HSP31 creates a synthetic lethality with protein import clogging under respiring conditions. We propose that Mfb1 functions to maintain mitochondrial protein import competency under clogging conditions, whereas Hsp31 plays an important role in protecting the cytosol against mPOS. Mutations in human mitochondria-associated F-box proteins (e.g., Fbxo7) and the human Hsp31 homolog, DJ-1, are known to cause early-onset Parkinson’s disease. Our work may improve the understanding of how these mutations cause neurodegeneration.
Project description:Mitochondrial biogenesis requires the import of many nuclear-encoded proteins across the Translocase of Outer Membrane (TOM), and the Translocase of Inner Membrane (TIM) 22 or 23 complexes. Protein import defects cannot only impair mitochondrial function but also cause mitochondrial Precursor Overaccumulation Stress (mPOS) in the cytosol. Recent studies showed that specific mutations in the nuclear-encoded Adenine Nucleotide Translocase 1 (ANT1) cause musculoskeletal and neurological diseases by clogging TOM and TIM22 and inducing mPOS. Here, we found that overexpression of MFB1, encoding the mitochondrial F-box protein 1, suppresses cell growth defect caused by a clogger allele of AAC2, the yeast homolog of Ant1. Disruption of MFB1 synergizes with a clogger allele of aac2 to inhibit cell growth. This is accompanied by increased retention of mitochondrial proteins in the cytosol, suggesting a severe defect in mitochondrial protein import. Proximity-dependent biotin identification (BioID) suggested that Mfb1 may interact with Tom22, a component of the TOM complex. Loss of MFB1 under clogging conditions activates genes encoding cytosolic chaperones including Hsp31. Interestingly, disruption of HSP31 creates a synthetic lethality with protein import clogging under respiring conditions. We propose that Mfb1 functions to maintain mitochondrial protein import competency under clogging conditions, whereas Hsp31 plays an important role in protecting the cytosol against mPOS. Mutations in human mitochondria-associated F-box proteins (e.g., Fbxo7) and the human Hsp31 homolog, DJ-1, are known to cause early-onset Parkinson’s disease. Our work may improve the understanding of how these mutations cause neurodegeneration.