Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

Oncogenesis in utero: fetal death due to acute myelogenous leukaemia with an MLL translocation.

The incidence of translocations involving the 11q23 gene MLL is markedly increased in leukaemias that occur in infants <1 year of age. Epidemiological and molecular data have demonstrated that at least some of these translocations occur in utero. In this report we describe a case of fetal death at 36 weeks of gestation. At autopsy the fetus was found to have widely disseminated acute myelogenous leukaemia (AML), FAB subtype M5. Molecular cytogenetic studies of nuclei recovered from paraffin-embedded tissue sections demonstrated that the leukaemic cells contained an MLL translocation. This is the first detailed report, to our knowledge, of fetal death due to acute leukaemia, and directly demonstrates oncogenesis in utero.

RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex.

To search genetically for additional components of the protein translocation apparatus of mitochondria, we have used low fidelity PCR mutagenesis to generate temperature-sensitive mutants in the outer membrane translocation pore component ISP42. A high copy number suppressor of temperature-sensitive isp42 has been isolated and sequenced. This novel gene, denoted ISP6, encodes a 61 amino acid integral membrane protein of the mitochondrial outer membrane, which is oriented with its amino-terminus facing the cytosol. Disruption of the ISP6 gene is without apparent effect in wild type yeast cells, but is lethal in temperature-sensitive isp42 mutants. Immunoprecipitation of the gene product, ISP42p, from mitochondria solubilized under mild conditions reveals a multi-protein complex containing ISP6p and ISP42p.

Interaction of heavy chain binding protein (BiP/GRP78) with adenine nucleotides.

Immunoglobulin heavy chain binding protein (BiP/GRP78) is a resident endoplasmic reticulum protein that binds tightly to a number of incompletely assembled or aberrant proteins. BiP also binds ATP and can be purified by ATP affinity chromatography. Here we show that an ATPase activity co-purifies with BiP prepared from canine pancreas. The BiP-associated ATPase has a high affinity for ATP but a low turnover number, suggesting a regulatory, rather than an enzymatic role. We also show that submicromolar levels of ATP or ADP decrease the rate of adsorption of [125I]BiP to nitrocellulose filters coated with protein or non-ionic detergents. In contrast, micromolar levels of AMP increase the rate of adsorption. Furthermore, ATP and ADP decrease the susceptibility of BiP to proteolytic degradation, whereas AMP was found to enhance degradation slightly. Adenine nucleotides may therefore induce or stabilize different conformations of BiP even when ATP hydrolysis does not occur.

Heavy-chain binding protein recognizes aberrant polypeptides translocated in vitro.

Immunoglobulin heavy-chain binding protein (BiP, GRP-78) associates tightly in the endoplasmic reticulum (ER) with newly synthesized proteins that are incompletely assembled, have mutant structures, or are incorrectly glycosylated. The function of BiP has been suggested to be to prevent secretion of incorrectly folded or incompletely assembled protein, to promote folding or assembly of proteins, or to solubilize protein aggregates within the ER lumen. Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translation-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulphide-bonded prolactin, but does not associate detectably with either glycosylated invertase or correctly disulphide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity in vitro to aberrantly folded or aberrantly glycosylated polypeptides, but not to all nascent chains as they are folding.