MULocDeep: A deep-learning framework for protein subcellular and suborganellar localization prediction with residue-level interpretation.

Prediction of protein localization plays an important role in understanding protein function and mechanisms. In this paper, we propose a general deep learning-based localization prediction framework, MULocDeep, which can predict multiple localizations of a protein at both subcellular and suborganellar levels. We collected a dataset with 44 suborganellar localization annotations in 10 major subcellular compartments-the most comprehensive suborganelle localization dataset to date. We also experimentally generated an independent dataset of mitochondrial proteins in Arabidopsis thaliana cell cultures, Solanum tuberosum tubers, and Vicia faba roots and made this dataset publicly available. Evaluations using the above datasets show that overall, MULocDeep outperforms other major methods at both subcellular and suborganellar levels. Furthermore, MULocDeep assesses each amino acid's contribution to localization, which provides insights into the mechanism of protein sorting and localization motifs. A web server can be accessed at http://mu-loc.org.

Estimating the number of protein molecules in a plant cell: protein and amino acid homeostasis during drought.

During drought stress, cellular proteostasis on the one hand and amino acid homeostasis on the other hand are severely challenged, because the decrease in photosynthesis induces massive proteolysis, leading to drastic changes in both the proteome and the free amino acid pool. Thus, we selected progressive drought stress in Arabidopsis (Arabidopsis thaliana) as a model to investigate on a quantitative level the balance between protein and free amino acid homeostasis. We analyzed the mass composition of the leaf proteome based on proteomics datasets, and estimated how many protein molecules are present in a plant cell and its subcellular compartments. In addition, we calculated stress-induced changes in the distribution of individual amino acids between the free and protein-bound pools. Under control conditions, an average Arabidopsis mesophyll cell contains about 25 billion protein molecules, of which 80% are localized in chloroplasts. Severe water deficiency leads to degradation of more than 40% of the leaf protein mass, and thus causes a drastic shift in distribution toward the free amino acid pool. Stress-induced proteolysis of just half of the 340 million RubisCO hexadecamers present in the chloroplasts of a single mesophyll cell doubles the cellular content of free amino acids. A major fraction of the amino acids released from proteins is channeled into synthesis of proline, which is a compatible osmolyte. Complete oxidation of the remaining fraction as an alternative respiratory substrate can fully compensate for the lack of photosynthesis-derived carbohydrates for several hours.

Combined genomic and proteomic approaches reveal DNA binding sites and interaction partners of TBX2 in the developing lung.

BACKGROUND: Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. METHODS: We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. RESULTS: We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. CONCLUSION: Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

The Plant Mitochondrial TAT Pathway Is Essential for Complex III Biogenesis.

Twin arginine translocation (TAT) pathways have been extensively studied in bacteria and chloroplasts for their role in membrane translocation of folded proteins. However, an increasing number of organisms have been found to contain mitochondria-located TAT subunits, including plant mitochondria, which contain TAT subunits, though in an unusual arrangement with only TatB and TatC subunits. To date, no confirmed function has been attributed to mitochondrial TAT pathways in any organism. Using a truncation mutant approach, we demonstrate that the plant mitochondrial TatB (MTTATB) is required for complex III biogenesis. More specifically, MTTATB performs at a late stage in complex III biogenesis, conveying the translocation of the C terminus of the Rieske FeS subunit back across the inner membrane. This work confirms that plant mitochondria retained a functional TAT pathway for the Rieske FeS translocation, most likely from the original mitochondrial ancestor. It is hypothesized that the original mitochondria contained a bacteria-derived TAT pathway required for at least the Rieske FeS translocation. In several eukaryotic lineages, this mitochondrial TAT pathway was lost and replaced by BCS1. Interestingly, plant mitochondria appear to assemble complex III in the same subunit order as yeast and mammals but in contrast use bacteria-like assembly factors for this process.

Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses.

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep tag in Arabidopsis (Arabidopsis thaliana) exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of >90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods. Among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.