Trevor Lithgow

The J‐protein family: modulating protein assembly, disassembly and translocation

DnaJ is a molecular chaperone and the prototypical member of the J‐protein family. J proteins are defined by the presence of a J domain that can regulate the activity of 70‐kDa heat‐shock proteins. Sequence analysis on the genome of Saccharomyces cerevisiae has revealed 22 proteins that establish four distinguishing structural features of the J domain: predicted helicity in segments IIV, precisely placed interhelical contact residues, a lysine‐rich surface on helix II and placement of the diagnostic sequence HPD between the predicted helices II and III. We suggest that this definition of the J‐protein family could be used for other genome‐wide studies. In addition, three J‐like proteins were identified in yeast that contain regions closely resembling a J domain, but in which the HPD motif is non‐conservatively replaced. We suggest that J‐like proteins might function to regulate the activity of bona fide J proteins during protein translocation, assembly and disassembly.

The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae

Transcription‐induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription‐associated recombination, as they both affect transcription elongation and hyper‐recombination in a concerted manner. Using a yeast strain that has the wild‐type THO2 gene replaced by one encoding a His6‐HA‐tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti‐Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Δ and tho2Δ cells, mft1Δ and thp2Δ cells show mitotic hyper‐ recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper‐recombination conferred by mft1Δ and thp2Δ is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram‐negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N‐terminal, periplasmic domain rich in POTRA repeats and a C‐terminal beta‐barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two‐partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.

Assembling the mitochondrial outer membrane

The general preprotein translocase of the outer mitochondrial membrane (TOM complex) transports virtually all mitochondrial precursor proteins, but cannot assemble outer-membrane precursors into functional complexes. A recently discovered sorting and assembly machinery (SAM complex) is essential for integration and assembly of outer-membrane proteins, revealing unexpected connections to mitochondrial evolution and morphology.

Functional cooperation of mitochondrial protein import receptors in yeast.

We have identified a 20 kDa yeast mitochondrial outer membrane protein (termed MAS20) which appears to function as a protein import receptor. We cloned, sequenced and physically mapped the MAS20 gene and found that the protein is homologous to the MOM19 import receptor from Neurospora crassa. MAS20 and MOM19 contain the sequence motif F‐X‐K‐A‐L‐X‐V/L, which is repeated several times with minor variations in the MAS70/MOM72 receptors. To determine how MAS20 functions together with the previously identified yeast receptor MAS70, we constructed yeast mutants lacking either one or both of the receptors. Deletion of either receptor alone had little or no effect on fermentative growth and only partially inhibited mitochondrial protein import in vivo. Deletion of both receptors was lethal. Deleting only MAS70 did not affect respiration; deleting only MAS20 caused loss of respiration, but respiration could be restored by overexpressing MAS70. Import of the F1‐ATPase beta‐subunit into isolated mitochondria was only partly inhibited by IgGs against either MAS20 or MAS70, but both IgGs inhibited import completely. We conclude that the two receptors have overlapping specificities for mitochondrial precursor proteins and that neither receptor is by itself essential.

RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin

The yeast cytosol contains multiple homologs of the DnaK and DnaJ chaperone family. Our current understanding of which homologs functionally interact is incomplete. Zuotin is a DnaJ homolog bound to the yeast ribosome. We have now identified the DnaK homolog Ssz1p/Pdr13p as zuotins partner chaperone. Zuotin and Ssz1p form a ribosome-associated complex (RAC) that is bound to the ribosome via the zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems, as the 1:1 complex is stable, even in the presence of ATP or ADP. In vitro, RAC stimulates the translocation of a ribosome-bound mitochondrial precursor protein into mitochondria, providing evidence for its chaperone-like effect on nascent chains. In agreement with the existence of a functional complex, deletion of each RAC subunit resulted in a similar phenotype in vivo. However, overexpression of zuotin partly rescued the growth defect of the Δssz1 strain, whereas overexpression of Ssz1p did not affect the Δzuo1 strain, suggesting a pivotal function for the DnaJ homolog.

Targeting of C‐Terminal (Tail)‐Anchored Proteins: Understanding how Cytoplasmic Activities are Anchored to Intracellular Membranes

A class of integral membrane proteins, referred to as ‘tail‐anchored proteins’, are inserted into phospholipid bilayers via a single segment of hydrophobic amino acids at the C‐terminus, thereby displaying a large functional domain in the cytosol. This membrane attachment strategy allows eukaryotic cells to position a wide range of cytoplasmic activities close to the surface of an intracellular membrane. Tail‐anchored proteins often, but not always, demonstrate a selective distribution to specific intracellular organelles. This membrane‐specific distribution is required for the large number of targeting proteins that are tail‐anchored, but may or may not be critical for the numerous tail‐anchored pro‐apoptotic and anti‐apoptotic proteins of the Bcl‐2 family. Recent work has begun to address the mechanism for targeting tail‐anchored proteins to their resident membranes, but questions remain. What targeting signals determine each proteins intracellular location? Are there receptors for these signals and, if so, how do they function? What steps are required to integrate tail‐anchored proteins into the phospholipid bilayers? In this Traffic Interchange, we summarise what is known about tail‐anchored proteins, and outline the areas that are currently under study.

The protein import receptor of mitochondria

Protein import into the mitochondria of Saccharomyces cerevisiae depends on two receptor subcomplexes composed of integral outer-membrane proteins. One subcomplex is the MAS37-MAS70 heterodimer, which preferentially recognizes the mature regions of precursor proteins associated with ATP-dependent cytosolic chaperones. The other subcomplex contains the acidic proteins MAS20 and MAS22, which recognize the positively charged targeting sequences of a wide variety of mitochondrial precursors. We propose that the two subcomplexes can act together as a single, multifunctional receptor that binds simultaneously to different regions of a precursor molecule.

Acidic receptor domains on both sides of the outer membrane mediate translocation of precursor proteins into yeast mitochondria.

Mitochondrial precursor proteins made in the cytosol bind to a hetero‐oligomeric protein import receptor on the mitochondrial surface and then pass through the translocation channel across the outer membrane. This translocation step is accelerated by an acidic domain of the receptor subunit Mas22p, which protrudes into the intermembrane space. This ‘trans’ domain of Mas22p specifically binds functional mitochondrial targeting peptides with a Kd of < 1 microM and is required to anchor the N‐terminal targeting sequence of a translocation‐arrested precursor in the intermembrane space. If this Mas22p domain is deleted, respiration‐driven growth of the cells is compromised and import of different precursors into isolated mitochondria is inhibited 3‐ to 8‐fold. Binding of precursors to the mitochondrial surface appears to be mediated by cytosolically exposed acidic domains of the receptor subunits Mas20p and Mas22p. Translocation of a precursor across the outer membrane thus appears to involve sequential binding of the precursors basic and amphiphilic targeting signal to acidic receptor domains on both sides of the membrane.