SUMMARY Mitochondria are essential for numerous cellular processes, yet hundreds of their proteins lack robust functional annotation. To reveal new functions for these proteins (termed MXPs) we assessed condition-specific protein-protein interactions for 50 select MXPs using affinity enrichment mass spectrometry. Our data connect MXPs to diverse mitochondrial processes, including multiple aspects of respiratory chain function. Building upon these observations, we validated C17orf89 as a complex I (CI) assembly factor. Disruption of C17orf89 markedly reduced CI activity, and its depletion is found in an unresolved case of CI deficiency. We likewise discovered that LYRM5 interacts with and deflavinates the electron transferring flavoprotein that shuttles electrons to coenzyme Q (CoQ). Finally, we identified a dynamic human CoQ biosynthetic complex involving multiple MXPs whose topology we map using purified components. Collectively, our data lend new mechanistic insight into respiratory chain-related activities and prioritize hundreds of additional interactions for further exploration of mitochondrial protein function.

SUMMARY The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation, but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

Coenzyme Q (CoQ) is an isoprenylated quinone that is essential for cellular respiration and is synthesized in mitochondria by the combined action of at least nine proteins (COQ1-9). Although most COQ proteins are known to catalyze modifications to CoQ precursors, the biochemical role of COQ9 remains unclear. Here, we report that a disease-related COQ9 mutation leads to extensive disruption of the CoQ protein biosynthetic complex in a mouse model, and that COQ9 specifically interacts with COQ7 through a series of conserved residues. Toward understanding how COQ9 can perform these functions, we solved the crystal structure of Homo sapiens COQ9 at 2.4 Å. Unexpectedly, our structure reveals that COQ9 has structural homology to the TFR family of bacterial transcriptional regulators, but that it adopts an atypical TFR dimer orientation and is not predicted to bind DNA. Our structure also reveals a lipid-binding site, and mass spectrometry-based analyses of purified COQ9 demonstrate that it associates with multiple lipid species, including CoQ itself. The conserved COQ9 residues necessary for its interaction with COQ7 comprise a surface patch around the lipid-binding site, suggesting that COQ9 might serve to present its bound lipid to COQ7. Collectively, our data define COQ9 as the first, to our knowledge, mammalian TFR structural homolog and suggest that its lipid-binding capacity and association with COQ7 are key features for enabling CoQ biosynthesis.biquinone, also known as coenzyme Q (CoQ), is a lipophilic, redox-active small molecule that is present in nearly every cellular membrane. CoQ is a critical component of the mitochondrial electron transport chain where it shuttles electrons from complexes I and II to complex III. In addition to its vital role in cellular respiration, CoQ is instrumental in cellular antioxidation, extracellular electron transport, and membrane rigidity (1).The de novo biosynthesis of CoQ in eukaryotes takes place in the mitochondrial matrix via the collective action of at least 10 proteins (COQ1-10; Fig. S1) (2). Mutations in these proteins can cause primary CoQ deficiency-a condition associated with cerebellar ataxia, kidney disease, isolated myopathy, and severe childhood-onset multisystemic disorders (3, 4). Alteration in CoQ levels has also been associated with significant life span extensions in organisms ranging from Saccharomyces cerevisiae to mice (5-7). In S. cerevisiae (2, 8, 9), and potentially in higher eukaryotes (10, 11), most of the COQ proteins form a biosynthetic complex on the matrix face of the inner mitochondrial membrane. Although the majority of these proteins catalyze chemical modifications to CoQ precursors, the biochemical functions for COQ4, 8, and 9 have yet to be elucidated (8, 12, 13). Recently, García-Corzo et al. developed a mouse harboring a truncated version of Coq9 (Coq9 R239X)-modeled after a similar mutation observed in a human patient-that causes an encephalomyopathy associated with CoQ deficiency (11,14). A hallmark feature of these mice is a decreas...

Background: Sigma-1 receptor (S1R) is an integral membrane ligand-binding receptor.Results: Gel filtration chromatography revealed oligomeric states that are stabilized by ligand binding and destabilized by mutations in the GXXXG integral membrane dimerization domain.Conclusion: Purified S1R binds small molecule ligands as an oligomer but not as a monomer.Significance: The results provide new insight into the function of S1R with ligands and proteins partners.

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intronexon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions ''antisense'' to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.higher plant ͉ transcriptome ͉ maskless array synthesizer

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.

The Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsis open reading frames into Escherichia coli expression vectors are presented along with an analysis of results from approximately 2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold-function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel Gateway protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the Gateway system.

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