Molecular architecture of the active mitochondrial protein gate.

Mitochondria fulfill central functions in cellular energetics, metabolism, and signaling. The outer membrane translocator complex (the TOM complex) imports most mitochondrial proteins, but its architecture is unknown. Using a cross-linking approach, we mapped the active translocator down to single amino acid residues, revealing different transport paths for preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel to recruit chaperones from the intermembrane space that guide the transfer of hydrophobic preproteins. The translocator contains three Tom40 ß-barrel channels sandwiched between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex exchanges with a dimeric isoform to assemble new TOM complexes. Dynamic coupling of α-helical receptors, ß-barrel channels, and chaperones generates a versatile machinery that transports about 1000 different proteins.

Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.

The human importin-ß family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-ß family carriers and then supplemented with a particular importin-ß family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-ß family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-ß. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.

Localization prediction and structure-based in silico analysis of bacterial proteins: with emphasis on outer membrane proteins.

In this chapter, we first discuss protein localization in bacteria and evaluate some localization prediction tools on an independent dataset. Next, we focus on ß-barrel outer membrane proteins (BOMPs), describing and evaluating new tools for BOMP detection and topology prediction. Finally, we apply general protein structure prediction methods on these proteins to show that the structure of most BOMPs in E. coli can be modeled reliably.

Characteristic amino acid distribution around segments unique to allergens.

Epitopes are located at the surface of allergens with which antibodies specifically bind. On the assumption that fragments unique to allergens have common, characteristic amino acid sequences, we compared the amino acid sequences of allergens with those of non-allergens. Segments around fragments unique to allergens showed wavelet-like distributions for several amino acids. Charged residues, alanine and glycine had positive peaks at the centre of the unique segments with small valleys on both sides, while aromatic residues, proline and cysteine showed the inverse distribution. Furthermore, the wavelet-like distribution of amino acids could be represented by a universal distribution function together with an index characterizing the intensity of the wavelet. Using the universal distribution function and the novel index of amino acids, we developed a simple method for extracting segments and fragments that are unique to allergens. The significance of the universal distribution function and the novel index is also discussed, by comparing the plot of the allergen-unique fragments index and dynamic fluctuation in the three dimensional structure of birch pollen allergen as both a single molecule and a complex with the corresponding antibody.

Vertebrate genomes code excess proteins with charge periodicity of 28 residues.

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.