Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 ϫ g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmicphase cells. In contrast, sterol ⌬ 24 -methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.

The yeast nascent polypeptide-associated complex (NAC) is encoded by two genes, EGD1 and EGD2, and is associated with cytoplasmic ribosomes. Yeast mutants lacking NAC (⌬egd2) are viable but suffer slight defects in the targeting of nascent polypeptides to several locations including the endoplasmic reticulum and mitochondria. If both NAC and Mft52p are missing from yeast cells, inefficient targeting of mitochondrial precursor proteins leads to defects in both mitochondrial function and morphology. We suggest that NAC provides a ribosomal environment for nascent mitochondrial targeting sequences to achieve secondary structure, thereby enhancing the efficiency of protein targeting.Proteins do not arrive at their final subcellular location by chance. Maintenance of correct protein targeting is crucial for cell function, and nascent polypeptides are ushered from ribosomes in the cytosol to their specific subcellular location by soluble targeting factors (1, 2). In the case of secreted proteins, it is clear that translation, targeting, and translocation across the membrane of the endoplasmic reticulum are intimately related processes (3). A soluble targeting factor, the signal recognition particle (SRP), binds the amino-terminal signal sequence on nascent secretory proteins as they emerge from the ribosome to initiate targeting to the endoplasmic reticulum and translocation across the membrane (4-7). The initial binding of SRP to ribosomes bearing nascent polypeptides is a regulated event. The nascent polypeptide-associated complex (NAC) sitting on the surface of mammalian ribosomes at the site of the emerging polypeptide chain can prevent binding of SRP to nascent chains without signal sequences and can hinder binding of vacant ribosomes to the endoplasmic reticulum because of its position on the surface of the ribosome (8-10).SRP binds exclusively to nascent polypeptides bearing secretion signals, but NAC initially contacts nascent polypeptides destined for many (and probably all) subcellular compartments. Both the ␣ and ␤ subunits of NAC can be crosslinked to nascent polypeptides destined for the cytosol, peroxisome, endoplasmic reticulum, and mitochondria (11). These in vitro assays suggest that NAC binds to nascent polypeptides before the specific factors that would regulate protein distribution and delivery. The sequence of events that determines specific delivery of secretory proteins to the endoplasmic reticulum has been well studied (3, 12), and we have begun to analyze the steps involved in specifically delivering precursor proteins from ribosomes to the mitochondria, including the role of the cytosolic targeting factor Mft52p (13).Here, we identify yeast NAC and find it associated with cytosolic ribosomes. Disruption of the EGD2 gene, encoding the ␣ subunit of the NAC heterodimer, leads to decreased targeting of proteins to the endoplasmic reticulum and mitochondria in vivo. In terms of mitochondrial targeting, yeast mutants lacking both NAC and Mft52p show synthetic mitochondrial defects: reduced ...

Ten mutants of the yeast

The ERG1 gene of Saccharomyces cerevisiae encodes squalene epoxidase, a key enzyme in the ergosterol pathway. ERG1 is an essential gene. Disruption of the gene with URA3 results in a lethal phenotype when cells are grown under aerobic conditions, even in the presence of ergosterol. However, cells are viable in the presence of ergosterol under anaerobic growth conditions during which ergosterol is taken up by cells. Physical and genetic mapping data reveal that ERG1 is located on the right arm of chromosome VII proximal to QCR9 at a distance of 14·6 cM from ADE3.

No abstract

Leishmania parasites cause a wide range of human diseases, are transmitted by sandflies and multiply in macrophages of the mammalian host. Within the alimentary track of the sandfly, the motile extracellular parasites divide and differentiate into infective metacyclic forms. Once inoculated into the mammalian host, metacyclics transform into non-motile, intracellular, infective amastigotes. The genomes of both New and Old World Leishmania species contain tandemly repeated SHERP genes, that code for a novel 6.2 kDa Small fiydrophilic associated protein. SHERP shows stage-regulated expression, with upregulation in metacyclics. However, whereas SHERP transcripts are detectable in both infective parasite stages, the SHERP protein is only present in metacyclics. Therefore, SHERP expression may be subject to translational control during the parasite life cycle. Indirect immunofluorescence and immunogold electron microscopy have demonstrated that SHERP localises to the ER and to the outer mitochondrial membrane. As the protein has no N-terminal signal sequence or ER-retention motifs, it is likely that SHERP is found on the cytoplasmic side of the ER and mitochondrial membranes. Cell fractionation experiments indicate that S H E W partitions exclusively into the cytoplasmic fraction, suggesting that interactions with the ER and mitochondrial membrane are unstable.We propose that this small, hydrophilic and acidic protein may form part of a multimeric complex that is involved in protein import into the ER and mitochondria of infective extracellular Leishmania 1247 Interactive protein partners of the enzyme lipoprotein lipase