To understand better the potential functional importance of the dual-lipid modifications found in a number of intracellular proteins of eukaryotes, we have examined how "tenaciously" various doubly-lipid-modified peptides, with sequences and lipid modifications reflecting those found in intracellular proteins, are anchored to lipid bilayer membranes. Fluorescent-labeled peptides bearing dual-lipid modifications were incorporated into large unilamellar egg phosphatidylcholine/phosphatidylglycerol vesicles, and the kinetics of spontaneous intervesicle transfer of the lipopeptides were monitored by a fluorescence-dequenching assay. Lipopeptides incorporating the stable "dual-anchor" motif -C(geranylgeranyl)XC(geranylgeranyl)-OMe found in several rab and homologous proteins exhibit very slow rates of interbilayer transfer (t1/2 > 50 h), as do lipopeptides incorporating myristoyl-GC(palmitoyl)X- and -C(palmitoyl)XC(farnesyl)-OMe motifs found in various src-related intracellular tyrosine kinases and G-protein alpha-subunits and in p21H-ras, respectively. Lipopeptides terminating in an unmethylated -C(geranylgeranyl)C(geranylgeranyl)-OH motif show somewhat greater but still very slow rates of spontaneous interbilayer transfer (t1/2 = ca. 10 h). Extrapolating from these results, we estimate that the rate of spontaneous desorption of the corresponding doubly-anchored proteins from membranes should be much slower than that of regulated, protein-mediated release (effected by binding to an "escort" protein or by de-S-acylation). As a result the intracellular distributions of these species (and particularly their targeting to specific intracellular membranes) are likely to be governed (and regulated) primarily by kinetic rather than thermodynamic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Summaryb-1,6-Glucan is an essential fungal-speci®c component of the Saccharomyces cerevisiae cell wall that interconnects all other wall components into a lattice. Considerable biochemical and genetic effort has been directed at the identi®cation and characterization of the steps involved in its biosynthesis. Structural studies show that the polymer plays a central role in wall structure, attaching mannoproteins via their glycosylphosphatidylinositol (GPI) glycan remnant to b-1,3-glucan and chitin. Genetic approaches have identi®ed genes that upon disruption result in b-1,6-glucan defects of varying severity, often with reduced growth or lethality. These gene products have been localized throughout the secretory pathway and at the cell surface, suggesting a possible biosynthetic route. Current structural and genetic data have therefore allowed the development of models to predict biosynthetic events. Based on knowledge of b-1,3-glucan and chitin synthesis, it is likely that the bulk of b-1,6-glucan polymer synthesis occurs at the cell surface, but requires key prior intracellular events. However, the activity of most of the identi®ed gene products remain unknown, making it unclear to what extent and how directly they contribute to the synthesis of this polymer. With the recent availability of new tools, reagents and methods (including genomics), the ®eld is poised for a convergence of biochemical and genetic methods to identify and characterize the biochemical steps in the synthesis of this polymer.

The E4orf4 protein of human adenovirus induces p53-independent apoptosis, a process that may promote cell death and viral spread. When expressed alone, E4orf4 kills transformed cells but not normal human cells. The only clear target of E4orf4 in mammalian cells is the Ba (B55) subunit of protein phosphatase 2A (PP2A), a member of one of three classes of regulatory B subunits. Here we report the eects of E4orf4 in Saccharomyces cerevisiae, which encodes two PP2A regulatory B subunits, CDC55 and RTS1, that share homology with mammalian B and B' subunits, respectively. E4orf4 expression was found to be toxic in yeast, resulting in the accumulation of cells in G2/M phase that failed to grow upon removal of E4orf4. E4orf4-expressing yeast also displayed an elongated cell morphology similar to cdc55 deletion strains. E4orf4 required CDC55 to elicit its eect, whereas RTS1 was dispensable. The recruitment of the PP2A holoenzyme by E4orf4 was entirely dependent on Cdc55. These studies indicate that E4orf4-induced apoptosis in mammalian cells and cell death in yeast require functional interactions with B-type subunits of PP2A. However, some inhibition of growth by E4orf4 was observed in the cdc55 strain and with an E4orf4 mutant that fails to interact with Cdc55, indicating that E4orf4 may possess a second Cdc55-independent function aecting cell growth. Oncogene (2001) 20, 5279 ± 5290.

Abstract. A variety of cysteine-containing, lipidmodified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid-modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S-acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC-and -CXC(farnesyl)-OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC-motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S-acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl-motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC-occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment.VARIETY of integral membrane proteins and reversibly membrane-associated proteins in eukaryotic cells exhibits posttranslational acylation on one or more cysteine residues, a modification that for a number of such proteins appears to be dynamic (6,39,42,45,81,82,84) and, in some cases, is modulated by physiological or pharmacological stimuli (15,31,45,46,63,86). Integral membrane proteins may be S-acylated either on cysteine residues near the cytoplasmic termini of transmembrane helixes (3,14,28,30,32,68,74) or on cytoplasmic cysteine residues more distant from a transmembrane helix (9,18,21,88). Among the reversibly membrane-associated proteins that undergo S-acylation are found a number of srchomologous nonreceptor tyrosine kinases, heterotrimeric G protein a subunits and monomeric G proteins (for reviews see 10,43,44,61,65,75). S-acylation has been shown to enhance the membrane association of a variety Address all correspondence to Dr. John R. Silvius, Department of Biochemistry, McGill University, Montrral, Qu6bec,...

We have isolated CaKRE9, a gene from Candida albicans, that is a functional homologue of the Saccharomyces cerevisiae KRE9 gene involved in ␤-1,6-glucan synthesis. Disruption of the CaKRE9 gene in C. albicans shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of CaKRE9 grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the CaKRE9 gene product is a potentially useful candidate as a target for fungal-specific drugs.