Mitochondria fulfill central functions in cellular energetics, metabolism and signaling. The outer membrane TOM40 complex imports virtually all mitochondrial proteins, however, its architecture and the molecular mechanisms of preprotein translocation are unknown. We mapped the active translocator with resolution down to single amino acid residues, discovering distinct transport paths for hydrophilic and hydrophobic preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel interior to recruit intermembrane space chaperones that guide the transfer of hydrophobic preproteins. The translocator possesses an intricate architecture with three Tom40 β-barrel channels sandwiched 2 between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex is in exchange with a dimeric isoform that is crucial for assembly of new TOM40 complexes. The dynamic coupling of α-helical receptors, β-barrel channels and chaperones generates a versatile machinery that manages transport of ~1,000 different proteins into mitochondria.One Sentence Summary: Architecture of the mitochondrial TOM40 entry gate identifies preprotein paths and the blueprint for its assembly.Main Text: Mitochondria are essential organelles in eukaryotic cells. They are pivotal for cellular ATP production, numerous metabolic pathways and regulatory processes, and programmed cell death. During evolution of eukaryotes, most genes for mitochondrial proteins were transferred to the nucleus. The proteins are synthesized as preproteins in the cytosol and imported back into mitochondria. Different classes of preproteins have been identified that either contain N-terminal targeting sequences (presequences) or internal targeting information in the mature part (1-3). The protein translocator of the outer membrane (TOM40 complex) functions as the main entry gate of mitochondria (1-3). Most of the >1,000 different mitochondrial proteins are imported by the TOM40 complex, followed by transfer to distinct intramitochondrial machineries specialized for individual classes of preproteins. Whereas the structurally known membrane protein complexes consist of either α-helical or β-barrel proteins, the TOM40 complex is composed of both α-helical and β-barrel integral membrane proteins. The complex consists of the channel-forming β-barrel protein Tom40 and six other subunits each containing single α-helical transmembrane (TM) segments: the receptor proteins Tom20, Tom22 and Tom70, and the small regulatory subunits Tom5, Tom6 and Tom7 (1-3). Tom40, Tom22 and the small Tom proteins form the TOM40 core complex, whereas Tom20 and Tom70 are more loosely associated with the complex. The molecular architecture of the complex has not been elucidated. It is thus unknown how α-helical and β-barrel membrane proteins can be combined into a functional complex and how diverse classes of preproteins can be transported by the same transmembrane channel.To define the archite...

The human importin-␤ 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. Most genetic processes, including chromosome replication and transcription, occur in the nucleus, and thus the selection of proteins that enter the nucleus and act there is crucial for cellular processes (1). During interphase, all nuclear proteins cross the nuclear envelope via nuclear pores, channels that constitute a selective permeability barrier for macromolecules. Only a fraction of proteins traverse these channels by means of free diffusion; most nuclear proteins are imported into or exported out of the nucleus with the assistance of importin-␤ (Imp-␤) 1 family proteins (Imp-␤s) (2, 3). Imp-␤s interact with the nuclear pore complex in a way that allows them to travel in and out of the nucleus. To drive cargo From the ‡Cellular Dynamics Laboratory, Advanced Science Institute, RIKEN, 2-1 Hirosawa,

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.

Proteins with a charge periodicity of 28 residues (PCP28) were found recently in the human proteome, and many of the annotated PCP28 were located in the nucleus (Ke et al., Jpn. J. Appl. Phys. 2007). The physical properties of the amino acid sequences were analyzed to detect the difference in the physicochemistry between the nuclear and cytoplasmic PCP28 and develop a software system to classify the two types of PCP28. A significant difference in the global parameters from the entire sequence and the local parameters around a segment with the highest positive charge density was found between the nuclear and cytoplasmic PCP28. The global classification score included the densities of proline and cysteine, and the negative charge density, while the local score included the symmetry of the charge distribution, the density of cysteine, and the positive charge density. A prediction system was developed using the global and local scores, which possessed a sensitivity and specificity of 92% and 88%, respectively. The mechanism of translocation of proteins to the nucleus is discussed using the parameters relevant to the predictive system.