A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

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. In this study we show that the plasma membrane [H+]ATPase of Saccharomyces cerevisiae is phosphorylated on multiple Ser and Thr residues in vivo. Phosphorylation occurs during the movement of newly synthesized ATPase from the ER to the cell surface, as revealed by the analysis of temperature-sensitive sec mutants blocked at successive steps of the secretory pathway. Two-dimensional phosphopeptide analysis of the ATPase indicates that, although most sites are phosphorylated at or before arrival in secretory vesi-T HE plasma membrane ATPase ofSaccharomyces cerevisiae is a proton pump that regulates cytoplasmic pH and provides the driving force for nutrient uptake (Serrano et al ., 1986, Serrano, 1989. Not surprisingly, in light of these critical physiologic functions, the [H+]ATPase is essential for cell viability. It has strong structural homology with other EX2 or P-type ATPases involved in maintaining ionic homeostasis, including the [Na+,K+] and Cat+ATPases of mammalian cells. Like other members of its class, the yeast ATPase has a catalytic subunit of -100 kD, hydrolyzes ATP via a transient aspartylphosphate intermediate, and is inhibited by low concentrations of vanadate. Sequence analysis of the PMAI gene encoding the yeast [H+]-ATPase predicts that the protein consists of a large central cytoplasmic domain containing putative sites for ATP binding and hydrolysis, anchored in the membrane by four hydrophobic segments at the NH2-terminal end and four to six hydrophobic segments at the COOH-terminal end .One approach taken in our laboratory to understanding structure-function relationships of the [H+]ATPase has been to study the acquisition of tertiary structure and activity during enzyme biogenesis . Recent work has established that newly synthesized [H+]ATPase becomes integrated into the endoplasmic reticulum membrane without cleavage of an NH2-terminal signal sequence, and is delivered to the cell surface via the secretory pathway (Holcomb et al., 1988) . In sec mutants blocked at discrete steps of the pathway, membrane biogenesis is prevented (Novick and Schekman, 1983 ;Tschopp et al ., 1984), and [H+]ATPase en route to the plasma membrane becomes trapped within the ER, Golgi compartment, or secretory vesicles (Brada and Schekman, 1988) . The ER and Golgi forms of the ATPase have not yet been examined for activity, but the enzyme accumulated in

In recent years there has been growing interest in the posttranslational regulation of P-type ATPases by protein kinasemediated phosphorylation. Pma1 H ؉ -ATPase, which is responsible for H ؉ -dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896 -918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14 N/ 15 N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H ؉ -ATPase. Pma1 H ϩ

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