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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...

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

SUMMARY CDP-diacylglycerol (CDP-DAG) is central of the phospholipid biosynthesis pathways in cells. A prevailing view is that only one CDP-DAG synthase named Cds1 is present in both the endoplasmic reticulum (ER) and mitochondrial inner membrane (IM), and mediates generation of CDP-DAG from phosphatidic acid (PA) and CTP. However, we demonstrate here by using yeast Saccharomyces cerevisiae as a model organism that Cds1 resides in the ER but not in mitochondria, and that Tam41, a highly conserved mitochondrial maintenance protein, directly catalyzes the formation of CDP-DAG from PA in the mitochondrial IM. We also find that inositol depletion by overexpressing an arrestin-related protein Art5 partially restores the defects of cell growth and CL synthesis in the absence of Tam41. The present findings unveil the missing step of the cardiolipin synthesis pathway in mitochondria as well as the flexibile regulation of phospholipid biosynthesis to respond to compromised CDP-DAG synthesis in mitochondria.

Mitochondrial protein import requires cooperation of the machineries called translocators in the outer and inner mitochondrial membranes. Here we analyze the interactions of Tom22, a multifunctional subunit of the outer membrane translocator TOM40 complex, with other translocator subunits such as Tom20, Tom40, and Tim50 and with substrate precursor proteins at a spatial resolution of the amino acid residue by in vivo and in organello site-specific photocross-linking. Changes in cross-linking patterns caused by excess substrate precursor proteins or presequence peptides indicate how the cytosolic receptor domain of Tom22 accepts substrate proteins and how the intermembrane space domain of Tom22 transfers them to Tim50 of the inner-membrane translocator.M itochondria, a powerhouse of eukaryotic cells, consist of 1,000-1,500 different proteins that are mainly synthesized in the cytosol and placed in four subcompartments, the outer and inner membranes and the aqueous intermembrane space (IMS) and matrix (1-3). Import and subsequent intramitochondrial sorting of mitochondrial proteins are mediated by the mitochondrial membrane-protein complexes called translocators. The translocator complexes in the outer and inner membranes are not tightly linked with each other, yet dynamically cooperate to achieve protein delivery to each mitochondrial subcompartment. Besides, translocator complexes are dynamic entities on their own, and alter their subunit-subunit interactions to work in demand for imported client proteins with different destinations (4-6). To understand the mechanism of protein transport by the mitochondrial import/sorting systems, it is essential to monitor and analyze dynamic interactions among the constituents of the systems, and in organello and in vivo are techniques providing sufficiently high spatial resolution.Most matrix proteins and some inner-membrane proteins are synthesized as precursor proteins with an amino-terminal cleavable presequence, which contains a mitochondrial targeting signal. Presequences generally have potentials to form a positively charged amphiphilic helical structure. The outer membrane translocator, the TOM40 complex, functions as an entry gate for most mitochondrial proteins. The yeast TOM40 complex is composed of the core complex consisting of Tom40, Tom22, Tom5, Tom6, and Tom7, and peripheral receptor subunits, Tom20 and Tom70. Tom20 is a general import receptor and anchored to the outer membrane by its N-terminal transmembrane (TM) segment. Tom22 spans the outer membrane by its central TM segment, with its N-terminal and C-terminal domains exposed to the cytosol and IMS, respectively (7,8). The cytosolic domains of Tom20 and Tom22 cooperate to form a presequence receptor site called the cis site to recognize mitochondrial targeting signals (9-12). After recognition by the receptor subunits of the TOM40 complex, presequence-containing precursor proteins move across the outer membrane through the β-barrel Tom40 channel (13-15), which is stabilized by Tom22 (16). Then the presequ...

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here, we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

The β-barrel assembly machinery (BAM) complex is essential for localization of surface proteins on bacterial cells, but the mechanism by which it functions is unclear. We developed a direct stochastic optical reconstruction microscopy (dSTORM) methodology to view the BAM complex in situ. Single-cell analysis showed that discrete membrane precincts housing several BAM complexes are distributed across the E. coli surface, with a nearest neighbor distance of ∼200 nm. The auxiliary lipoprotein subunit BamB was crucial for this spatial distribution, and in situ crosslinking shows that BamB makes intimate contacts with BamA and BamB in neighboring BAM complexes within the precinct. The BAM complex precincts swell when outer membrane protein synthesis is maximal, visual proof that the precincts are active in protein assembly. This nanoscale interrogation of the BAM complex in situ suggests a model whereby bacterial outer membranes contain highly organized assembly precincts to drive integral protein assembly.

Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.