Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events that occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cellbased assays that monitor changes in cell proliferation, migration and morphogenesis in MCF10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

Yeast and human glycosylphosphatidylinositol (GPI) precursors differ in the extent to which a fourth mannose is present as a side branch of the third core mannose. A fourth mannose addition to GPIs has scarcely been detected in studies of mammalian GPI synthesis but is an essential step in the Saccharomyces cerevisiae pathway. We report that human SMP3 encodes a functional homolog of the yeast Smp3 GPI fourth mannosyltransferase. Expression of hSMP3 in yeast complements growth and biochemical defects of smp3 mutants and permits in vivo mannosylation of trimannosyl (Man 3 )-GPIs. Immunolocalization shows that hSmp3p resides in the endoplasmic reticulum in human cells. Northern analysis of mRNA from human tissues and cell lines indicates that hSMP3 is expressed in most tissues, with the highest levels in brain and colon, but its mRNA is nearly absent from cultured human cell lines. Correspondingly, increasing expression of hSMP3 in cultured HeLa cells causes abundant formation of three putative tetramannosyl (Man 4 )-GPIs. Our data indicate that hSmp3p functions as a mannosyltransferase that adds a fourth mannose to certain Man 3 -GPIs during biosynthesis of the human GPI precursor, and suggest it may do so in a tissue-specific manner.Glycosylphosphatidylinositols (GPIs) 1 are essential glycolipids synthesized by all eukaryotes. Many GPIs become covalently attached to the carboxyl termini of various secretory proteins and serve to anchor them to the exterior face of the plasma membrane (1, 2). Others remain protein-free and are distributed in the membranes of major cellular organelles and the plasma membrane (3, 4). GPIs are synthesized in the endoplasmic reticulum (ER) by stepwise addition of components to phosphatidylinositol. The end product of GPI synthesis is a "complete precursor" (5) that is substrate for the GPI transamidase complex that attaches it to proteins. Many of the steps and enzymes of GPI precursor assembly are conserved between humans and Saccharomyces cerevisiae and produce precursors with a common core structure of EthN-PO 4 -6Man␣1,2Man␣1,6Man␣1,4GlcN␣1,6Ins-PO 4 -lipid. In both pathways, the glycan portion of the GPI may be modified further with side branching ethanolamine phosphate (EthN-P) moieties on the first and second mannoses (6 -19).A notable difference in GPI structure between yeast and mammals is the extent to which a fourth mannose (Man-4) is present as a ␣1,2-linked side branch of the third mannose (Man-3). In S. cerevisiae, late stage intermediates in GPI precursor synthesis (17, 19 -21), the presumed GPI transamidase substrates (5), and protein-bound GPIs (22) all contain four mannoses. Addition of Man-4 to trimannosyl-GPIs (Man 3 -GPIs) by the essential Smp3 mannosyltransferase is a mandatory step in yeast GPI precursor assembly which precedes the addition of the terminal EthN-P to Man-3 through which the GPI is ultimately attached to protein (23). Thus, it is probable that all yeast GPI transamidase substrates bear four mannoses. Conversely, studies of the synthesis of mammal...

Yeast mcd4-174 mutants are blocked in glycosylphosphatidylinositol (GPI) anchoring of protein, but the stage at which GPI biosynthesis is interrupted in vivo has not been identified, and Mcd4p has also been implicated in phosphatidylserine and ATP transport. We report that the major GPI that accumulates in mcd4-174 in vivo is Man(2)-GlcN-(acyl-Ins)PI, consistent with proposals that Mcd4p adds phosphoethanolamine to the first mannose of yeast GPI precursors. Mcd4p-dependent modification of GPIs can partially be bypassed in the mcd4-174/gpi11 double mutant and in mcd4Delta; mutants by high-level expression of PIG-B and GPI10, which respectively encode the human and yeast mannosyltransferases that add the third mannose of the GPI precursor. Rescue of mcd4Delta; by GPI10 indicates that Mcd4p-dependent addition of EthN-P to the first mannose of GPIs is not obligatory for transfer of the third mannose by Gpi10p.