Roland Lill

The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins

The ATPase activity of SecA is stimulated by E. coli plasma membrane vesicles bearing SecY protein and a precursor protein such as proOmpA. This activity is termed translocation ATPase. Liposomes alone can also stimulate SecA ATPase, but membrane proteins block this stimulation in native inner membranes. We define the stimulation of SecA ATPase by lipid as SecA/lipid ATPase. SecA/lipid ATPase, translocation ATPase, and translocation into inner membrane vesicles require acidic phospholipids, suggesting an underlying unity of mechanism. ProOmpA and ATP stabilize liposome-bound SecA. Full SecA/lipid ATPase activity and stability are also seen when a mixture of a leader peptide and either OmpA or maltose binding protein (MBP) are added instead of proOmpA, while neither the leader peptide alone nor OmpA or MBP suffice. Cytosolic proteins in conjuction with a leader peptide are less active in this reaction, indicating that liposome-bound SecA protein recognizes both leader and mature domains.

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation‐coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation‐competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY‐thermosensitive strain which had been incubated at the non‐permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8‐azido‐ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre‐secretory proteins across the membrane.

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli‐‐SecB, trigger factor and GroEL‐‐have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed ‘chaperones’. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation‐ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

Activation of the iron regulon by the yeast Aft1/Aft2 transcription factors depends on mitochondrial but not cytosolic iron-sulfur protein biogenesis.

Two transcriptional activators, Aft1 and Aft2, regulate iron homeostasis in Saccharomyces cerevisiae. These factors induce the expression of iron regulon genes in iron-deficient yeast but are inactivated in iron-replete cells. Iron inhibition of Aft1/Aft2 is abrogated in cells defective for Fe-S cluster biogenesis within the mitochondrial matrix (Chen, O. S., Crisp, R. J., Valachovic, M., Bard, M., Winge, D. R., and Kaplan, J. (2004) J. Biol. Chem. 279, 29513–29518). To determine whether iron sensing by Aft1/Aft2 requires the function of the mitochondrial Fe-S export and cytosolic Fe-S protein assembly systems, we evaluated the expression of the iron regulon in cells depleted of glutathione and in cells depleted of Atm1, Nar1, Cfd1, and Nbp35. The iron regulon is induced in cells depleted of Atm1 with Aft1 largely responsible for the induced gene expression. Aft2 is activated at a later time in Atm1-depleted cells. Likewise, the iron regulon is induced in cells depleted of glutathione. In contrast, repression of NAR1, CFD1, or NBP35 fails to induce the iron regulon despite strong inhibition of cytosolic/nuclear Fe-S protein assembly. Thus, iron sensing by Aft1/Aft2 is not linked to the maturation of cytosolic/nuclear Fe-S proteins, but the mitochondrial inner membrane transporter Atm1 is important to transport the inhibitory signal. Although Aft1 and Aft2 sense a signal emanating from the Fe-S cluster biogenesis pathway, there is no indication that the proteins are inhibited by direct binding of an Fe-S cluster.

Phospholipid composition of highly purified mitochondrial outer membranes of rat liver and Neurospora crassa. Is cardiolipin present in the mitochondrial outer membrane

Isolated mitochondrial outer membrane vesicles (OMV) are a suitable system for studying various functions of the mitochondrial outer membrane. For studies on mitochondrial lipid import as well as for studies on the role of lipids in processes occurring in the outer membrane, knowledge of the phospholipid composition of the outer membrane is indispensable. Recently, a mild subfractionation procedure was described for the isolation of highly purified OMV from mitochondria of Neurospora crassa (Mayer, A., Lill, R. and Neupert, W. (1993) J. Cell Biol. 121, 1233-1243). This procedure, which consists of swelling and mechanical disruption of mitochondria followed by two steps of sucrose density gradient centrifugation, was adapted for the isolation of OMV from rat liver mitochondria. Using the appropriate enzyme markers it is shown that the resulting OMV are obtained in a yield of 25%, and that their purity is superior to that of previous OMV preparations. Analysis of the phospholipid composition of the OMV showed that phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol are the major phospholipid constituents, and that cardiolipin is only present in trace amounts. The phospholipid composition is very similar to that of the highly purified OMV from mitochondria of Neurospora crassa, although the latter still contain a small amount of cardiolipin.

Mitochondrial protein import: Reversible binding of the presequence at the trans side of the outer membrane drives partial translocation and unfolding

The mechanism of translocation of matrix-targeted, cleavable preproteins across the mitochondrial outer membrane was studied using purified outer membrane vesicles. The N-terminal presequence interacts in a sequential and reversible fashion with two specific binding sites. The first one is provided by protease-sensitive receptors on the surface of the membrane (cis site); the second one is located at the inner face of the outer membrane (trans site). Binding to the trans site drives translocation of the N-terminal portion of the preprotein and, at the same time, unfolding of its mature part. We suggest that the outer membrane protein import machinery forms a translocation channel that permits reversible sliding of preproteins and prevents their lateral aggregation in the membrane. Although translocation can be initiated by the outer membrane, its completion requires coupling to the energetic system of the inner membrane. Our data assign an essential role to the presequence, not only for efficient targeting, but also for the translocation step.

Mitochondrial and cytosolic branched-chain amino acid transaminases from yeast, homologs of the myc oncogene-regulated Eca39 protein

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea‐treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea‐treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle

n Abstractn n We have isolated large amounts of E. coli outer-membrane protein A precursor (proOmpA). Purified proOmpA is active in membrane assembly, and this assembly is saturable with respect to the precursor protein. A proOmpA-Sepharose matrix allows affinity isolation of trigger factor, a soluble, 63,000 dalton monomeric protein that stabilizes proOmpA in assembly competent form. Comparison of trigger factors amino-terminal sequence with those in a computer data bank and with those encoded by sec genes, as well as groEL and heat shock gene dnaK, suggests that trigger factor is encoded by a previously undescribed gene. Trigger factor and proOmpA form a 1:1 complex that can be isolated by gel filtration. Purified canine signal recognition particle (SRP) can also stabilize proOmpA for membrane insertion. This post-ribosomal activity of SRP suggests a unifying theme in protein translocation mechanisms.n n

The trigger factor cycle includes ribosomes, presecretory proteins, and the plasma membrane

Trigger factor is a soluble, 63,000 dalton protein of E. coli that stabilizes proOmpA, the precursor form of a major outer-membrane protein, in a conformation competent for in vitro membrane assembly. There is approximately one trigger factor molecule bound to each 70S ribosome isolated from cell extracts in physiological buffers. Trigger factor dissociates from ribosomes in 1.5 M LiCl and reassociates with salt-washed ribosomes in low-salt buffer. Binding is exclusively to the 50S (large) subunit, known to contain the exit domain for nascent polypeptide chains. In addition to its associations with proOmpA and ribosomes, excess trigger factor can compete with the proOmpA-trigger factor complex for a limited number of membrane sites that are essential for translocation of proOmpA. These data suggest a model of trigger factor cycling between the cytoplasm, the ribosome, presecretory proteins, and membrane receptor proteins.