Based on the prevalence of modular protein domains, such as Src homology domain 3 and 2 (SH3 and SH2), among important signaling molecules, we have sought to identify new SH3 domain-containing proteins. However, modest sequence similarity among these domains restricts the use of DNA-based methods for this purpose. To circumvent this limitation, we have developed a functional screen that permits the rapid cloning of modular domains based on their ligand-binding activity. Using operationally defined SH3 ligands from combinatorial peptide libraries, we screened a series of mouse and human cDNA expression libraries. We found that 69 of the 74 clones isolated encode at least one SH3 domain. These clones encode 18 different SH3-containing proteins, 10 of which have not been described previously. The isolation of entire repertoires of modular domain-containing proteins will prove invaluable in genome analysis and in bringing new targets into drug discovery programs.

A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-␣, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.The recognition and elucidation in recent years of various modular protein domains, along with their specific peptide ligands, have spawned a remarkable progress in our understanding of their role in signal transduction and other fundamental cellular processes (1, 2). Analysis of the SH (Src homology) domains, SH2 and SH3, present in a wide variety of proteins involved in cellular signaling and transformation has been particularly fruitful. The ligand specificity of many different SH2 and SH3 domains has been defined using combinatorial peptide libraries. SH2 domains bind with high affinity to phosphotyrosine residues within a specific sequence context (3). In contrast, SH3 domains bind to proline-rich peptides that share a conserved PXXP motif (4, 5).A newly described protein module, termed the WW domain, has been reported (6 -8). The WW domain consists of 35-40 amino acids and is characterized by four well conserved aromatic residues, two of which are tryptophan. The secondary structure of the WW domain has recently been determined and consists of a slightly bent three-stranded antiparallel ␤-sheet (9). This domain has been reported in a wide variety of proteins of yeast, nematode, and vertebrate origin, including Rsp5, Yesassociated protein (YAP), 1 human and murine Nedd-4, FE65, Pin1, and a human . Although the precise physiological role of the WW domain remains undetermined, its presence in diverse proteins involved in signaling, regulatory, and cytoskeletal functions, as well as its rapidly emerging role in signaling mechanisms that underlie several human diseases, clearly underscores its importance (15, 16). Two ligand proteins for the YAP WW domain, WBP-1...

Abstract. The distribution of mitochondria to daughter cells is an essential feature of mitotic cell growth, yet the molecular mechanisms facilitating this mitochondrial inheritance are unknown. We have isolated mutants of Saccharomyces cerevisiae that are temperature-sensitive for the transfer of mitochondria into a growing bud. Two of these mutants contain single, recessive, nuclear mutations, mdm/and mdm2, that cause temperature-sensitive growth and aberrant mitochondrial distribution at the nonpermissive temperature. The absence of mitochondria from the buds of mutant cells was confirmed by indirect iramunofluorescence microscopy and by transmission electron microscopy. The mdm/lesion also retards nuclear division and prevents the transfer of nuclei into the buds. Cells containing the mdm2 mutation grown at the nonpermissive temperature sequentially form multiple buds, each receiving a nucleus but no mitochondria. Neither mdm/or mdm2 affects the transfer of vacuolar material into the buds or causes apparent changes in the tubulin-or actin-based cytoskeletons. The mdm/ and mdm2 mutations are cell-cycle specific, displaying an execution point in late G1 or early S phase.

Abstract. The mdm/ mutation causes temperaturesensitive growth and defective transfer of nuclei and mitochondria into developing buds of yeast cells at the nonpermissive temperature. The MDM1 gene was cloned by complementation, and its sequence revealed an open reading frame encoding a potential protein product of 51.5 kD. This protein displays amino acid sequence similarities to hamster vimentin and mouse epidermal keratin. Gene disruption demonstrated that MDM1 is essential for mitotic growth. Antibodies against the MDM1 protein recognized a 51-kD polypeptide that was localized by indirect immunofluorescence to a novel pattern of spots and punctate arrays distributed throughout the yeast cell cytoplasm. These structures disappeared after shifting mdm/mutant cells to the nonpermissive temperature, although the cellular level of MDM1 protein was unchanged. Affinitypurified antibodies against MDM1 also specifically recognized intermediate filaments by indirect immunofluorescence of animal cells. These results suggest that novel cytoplasmic structures containing the MDM1 protein mediate organelle inheritance in yeast.

Intermediate filaments are abundant cytoskeletal components whose specific cellular functions are poorly understood. The Saccharomyces cerevisiae protein MDM1 displays structure and solubility properties that are similar to those of intermediate filament proteins of animal cells. Yeast cells that have a mutant form of MDM1 exhibit temperature-sensitive growth and defective transfer of nuclei and mitochondria to daughter cells during incubation at the nonpermissive temperature of 37 degrees C. The purified, wild-type MDM1 protein readily forms 10-nanometer-wide filaments at either 4 degrees C or 37 degrees C. In contrast, the purified, mutant protein forms filaments at 4 degrees C but fails to form such structures at 37 degrees C. These results suggest that intermediate filament proteins are universal components of eukaryotic cells.

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