The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast.

We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors.

Yeast mitochondria lacking the two import receptors Mas20p and Mas70p can efficiently and specifically import precursor proteins.

Mas20p and Mas70p are integral proteins of the yeast mitochondrial outer membrane that appear to function as receptors for precursor proteins imported from the cytosol. Loss of either receptor alone does not block import or kill the cells, but deletion of Mas20p causes loss of respiration (Ramage, L., Junne, T.

The protein product of the oncogene bcl-2 is a component of the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane.

The protein product of the oncogene bcl-2 is a potent inhibitor of apoptotic cell death. The Bcl-2 protein has variously been reported to reside in the nuclear envelope and endoplasmic reticulum or exclusively in the inner membrane of mitochondria. We therefore undertook a detailed analysis of the intracellular distribution of Bcl-2 by immunofluorescence, immunogold electron microscopy, and subcellular fractionation in three mouse cell lines expressing a human bcl-2 transgene and measured its importation into isolated mitochondria.

An Escherichia coli gene showing a potential ancestral relationship to the genes for the mitochondrial import site proteins ISP42 and MOM38.

An ORF (OrfT) of 1911 base pairs, upstream of the hip operon in Escherichia coli at map position 33.82 has been identified. The protein encoded by this sequence is predicted to have a molecular mass of 68,249 Da and the carboxyterminal 276 residues shows 26.

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.

A constitutive form of heat-shock protein 70 is located in the outer membranes of mitochondria from rat liver.

HSP73, the constitutive form of heat-shock protein 70, has been implicated in the translocation of preproteins across the mitochondrial membranes, being required for maintaining mitochondrial preproteins in an import competent conformation. Here we report that highly purified mitochondrial outer membranes contain a protein indistinguishable from HSP73 as a tightly associated peripheral component of the membrane. This membrane form of HSP73 was photolabelled with [alpha-32P]ATP and could be released from the outer membrane with sodium carbonate, but not after incubation of the membranes with salt or with ATP.

Do cytosolic factors prevent promiscuity at the membrane surface?

From the point of view of a preprotein, escaping from the cytosol into a specific organelle must seem an arduous, almost impossible task. How is it that preproteins resist the temptation to fold prematurely, to avoid the multiple membrane surfaces in the cell, and manage instead to enter only the translocation apparatus of a single organelle?

Prechaperonin 60 and preornithine transcarbamylase share components of the import apparatus but have distinct maturation pathways in rat liver mitochondria.

Mitochondrial preornithine transcarbamylase (p-OTC) and premalate dehydrogenase (p-MDH) are the only two matrix-located preproteins so far identified for which the proteolytic processing in vitro requires the formation of genuine processing intermediates, i-OTC and i-MDH, respectively. To establish the processing of other preproteins during import with respect to the two-step processing of p-OTC and p-MDH, the chelators EDTA and 1,10-phenanthroline were used to study the import and processing of rat prechaperonin 60 (p-cpn60) and p-OTC by mitochondria from four cpn60-containing organs. We found no evidence for a secondary processing step in the maturation of p-cpn60, but a clear requirement for two-step processing of p-OTC, even in three organs which do not contain ornithine transcarbamylase.

Identification of a GTP-binding protein in the contact sites between inner and outer mitochondrial membranes.

Whilst investigating whether GTP hydrolysis may be required for the import of preproteins into mitochondria we have found that a GTP-binding protein is located at the contact sites between mitochondrial inner and outer membranes. When mitochondrial outer membranes purified from rat liver were UV-irradiated in the presence of [alpha-32P]GTP, a 52 kDa protein was radiolabelled, whereas [alpha-32P]ATP did not label this protein. GTP-binding proteins were also labelled in the cytosolic and microsomal fractions, but the 52 kDa protein was concentrated in mitochondrial membranes and was the only protein specifically labelled by GTP in these membranes.

High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system.

The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells.

Primary structure of mammalian ribosomal protein S6.

Ribosomal protein S6 was isolated from rat liver ribosomes by reversed-phase high-performance liquid chromatography (HPLC) and subjected to cyanogen bromide and proteolytic cleavages. The cleavage fragments were resolved by HPLC and sequenced by automated Edman degradation. The overall amino acid sequence of S6 (249 residues) was determined by alignment of the overlapping sequences of selected cyanogen bromide, chymotryptic, tryptic, and clostripain cleavage fragments.

Pharmaco-ontogeny of reward: enhancement of self-stimulation by D-amphetamine and cocaine in 3- and 10-day-old rats.

Electrodes were implanted in various forebrain sites in 3- and 10-day-old rat pups that were allowed to nudge a pole for electrical stimulation of those areas. After 5 h pups were injected with amphetamine, cocaine or saline and the change in responding noted. Both stimulants, at relatively high doses, enhanced responding on the pole that produced stimulation to a greater extent than responding on a non-active pole.

Self-stimulation in 7- and 10-day-old rats.

Recently, it has been shown that the infant rat exhibits learned behaviors characteristic of the adult. With a modified self-stimulation paradigm, this study explored whether 7- and 10-day-old rat pups could learn a discriminated operant to obtain direct electrical stimulation in neural sites that support self-stimulation in adults. By nudging one of two response manipulanda, at two ages (7 and 10 days) and temperatures (22 and 35 degrees C), pups self-stimulated with electrodes implanted in a variety of forebrain sites, including the prefrontal cortex, bed nucleus of the stria terminalis, medial nucleus of the amygdala, and the medial forebrain bundle.

Effect of age on benzodiazepine-induced behavioural convulsions in rats.

The benzodiazepines are a class of drugs used to alleviate anxiety. As such they constitute one of the most commonly prescribed compounds, due in part to their efficacy and safety. The physiological effect of these drugs is probably through interactions with a low affinity benzodiazepine binding site and two (types 1 and 2) higher affinity sites.

A method for stereotaxic implantation in neonatal rats.

Presented are methods of implanting chronically indwelling electrodes or cannulas into brain infant rats. Anesthesized pups are restrained in a flexible alginate mold that is place in the stereotaxic instrument. Details of electrode implant are given.