The retinoblastoma protein {pll0 nB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing [unction. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind pll0 RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-la2), which differs from the originally defined PP-lc~ by an amino-terminal l 1-amino-acid insert. In vitro-binding assays demonstrated that PP-lc~ isoforms preferentially bind the hypophosphorylated form of p ll0 RB. Moreover, similar pll0 RB sequences are required for binding PP-lc~2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early Gv The implications of these findings on the regulation of both proteins are discussed.

pep12/vps6 mutants of Saccharomyces cerevisiae are defective in delivery of soluble vacuolar hydrolases to the vacuole. Morphological analysis by electron microscopy revealed that pep12 cells accumulate 40- to 50-nm vesicles. Furthermore, pep12 cells have enlarged vacuoles characteristic of class D pep/vps mutants. PEP12 encodes a protein of 288 amino acids that has a C-terminal hydrophobic region and shares significant sequence similarity with members of the syntaxin protein family. These proteins appear to participate in the docking and fusion of intracellular transport vesicles. Pep12p is the first member of the syntaxin family to be implicated in transport between the Golgi and the vacuole/lysosome. Pep12p-specific polyclonal antisera detected a 35-kDa protein that fractionated as an integral membrane protein. Subcellular fractionation experiments revealed that Pep12p was associated with membrane fractions of two different densities; the major pool (approximately 90%) of pep12p may associate with the endosome, while a minor pool (approximately 10%) cofractionated with the late Golgi marker Kex2p. These observations suggest that Pep12p may mediate the docking of Golgi-derived transport vesicles at the endosome.

Mutations in top, the structural gene for Escherichia coli DNA topoisomerase I, have been identified and mapped at 28 min on the chromosome, near cysB. Strains carrying deletions of the top gene are viable. The top mutations, however, do exert pleiotropic effects on transcription and transposition. Mutants lacking DNA topoisomerase I have a more rapid rate of induction and a higher level of catabolite-sensitive enzymes including tryptophanase and -galactosidase. This general activation of transcription by top mutations can be attributed to an increase in the negative superhelicity of the DNA in vivo when the topoisomerase activity is abolished. The frequency of transposition of Tn5, a transposon carrying kanamycin resistance, is decreased by a factor of 40 or more in top mutants. A direct or indirect role of the topoisomerase in transposition is discussed. The transposition frequency ofTn3, however, is not dependent on top. Based on the studies of the E. coli top mutants, it appears that the supX gene, which was originally studied in SalmoneUa typhimurium [Dubnau, E. & Margolin, P. (1972) Mol. Gen. Genet. 117, 91-112] is likely to be the structural gene for DNA topoisomerase I.Escherichia coli DNA topoisomerase I, also known as w protein, has been the subject of extensive studies (for a review, see ref.1). The enzyme is a single polypeptide ofabout 110,000 daltons. In vitro it catalyzes the relaxation of negatively supercoiled DNA (2), the knotting and unknotting of single-stranded DNA rings (3), the linking of single-stranded DNA rings of complementary sequences into intertwined duplex rings (4), and the catenation and decatenation of duplex DNA rings when at least one member of a pair of participating duplex rings has a singlechain scission (5). The catalysis of these topological isomerization reactions by the enzyme is believed to involve transient single-stranded breakage of DNA phosphodiester bonds (1).In contrast to the extensive in vitro studies, there has been little work on the functions of the enzyme in vivo. We have therefore undertaken the isolation and characterization of mutants deficient in DNA topoisomerase I activity. In this communication, we report the identification of a number of mutations in the structural gene for the enzyme and the genetic mapping ofthis gene, which we have termed top, on the E. coli chromosome. Roles of the enzyme in the regulation of transcription ofa number ofoperons and in the transposition of several transposons are also implicated based on our initial characterization of the mutants.MATERIALS AND METHODS Bacterial Strains. All strains used were E. coli K-12 derivatives. The strain used to produce the collection oftemperaturesensitive mutants was PA3092 F-thr leu thi argH thyA his trp lacYl mtl xyl malA mel tonA str supE. Two mutants from this collection were found to have top mutations, JE10010 topl0 and JE10250 top250 (6). Other strains used include: P4X8 Hfr met; KV385 met lacYl xyl mtl str topl0; PLK831 F-trpE pyrF gal-25 nirA strA195; JTT1, a trp+ top+...

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The Saccharomyces cerevisiae PEP3 gene was cloned from a wild-type genomic library by complementation of the carboxypeptidase Y deficiency in a pep3-12 strain. Subclone complementation results localized the PEP3 gene to a 3.8-kb DNA fragment. The DNA sequence of the fragment was determined; a 2,754-bp open reading frame predicts that the PEP3 gene product is a hydrophilic, 107-kDa protein that has no significant similarity to any known protein. The PEP3 predicted protein has a zinc finger (CX2CX13CX2C) near its C terminus that has spacing and slight sequence similarity to the adenovirus Ela zinc finger. A radiolabeled PEP3 DNA probe hybridized to an RNA transcript of 3.1 kb in extracts of log-phase and diauxic lag-phase cells. CeUls bearing pep3 deletion/disruption alleles were viable, had decreased levels of protease A, protease B, and carboxypeptidase Y antigens, had decreased repressible alkaline phosphatase activity, and contained very few normal vacuolelike organelles by fluorescence microscopy and electron microscopy but had an abundance of extremely smal vesicles that stained with carboxyfluorescein diacetate, were severely inhibited for growth at 37C, and were incapable of sporulating (as homozygotes). Fractionation of ceUs expressing a bifunctional PEP3::SUC2 fusion protein indicated that the PEP3 gene product is present at low abundance in both log-phase and stationary cels and is a vacuolar peripheral membrane protein. Sequence identity established that PEP3 and VPS18 (J. S. Robinson, T. R. Graham, and S. D. Emr, Mol. Cell. Biol. 11:5813-5824, 1991) are the same gene.The vacuole in Saccharomyces cerevisiae is a polymorphic organelle that has similarities both to vacuoles in plants and to lysosomes in animal cells. Like lysosomes, yeast vacuoles are relatively acidic organelles that contain a number of proteases and other hydrolases that have relatively broad substrate specificities. Mutants that lack particular vacuolar proteases have been useful for determining the functions of specific enzymes (23,25). One search for protease-deficient mutants unexpectedly identified 16 genes, mutations in any of which gave rise to deficiencies of two or three different vacuolar hydrolase activities (PEP genes) (21). Because pep mutations are pleiotropic, the phenotypes of the mutants provide very limited information about the primary functions of the PEP gene products. At the time the genes were identified, several general hypotheses were advanced to account for the pleiotropy of the mutations: that (i) these are regulatory mutations, (ii) the mutations cause changes in the structure of the compartments containing the enzymes, or (iii) the mutations alter components of the system that places the enzymes in the compartments (21). Thus, the PEP genes appeared to provide access to cellular "capital equipment" common to the expression of more than one vacuolar protease, but it was not clear in biochemical terms what type of equipment that might be. All three of the mechanisms mentioned above (or others not thought of)...

The gene topA of Escherichia coli that encodes for DNA topoisomerase I has been cloned by a combination of genetic and radioimmunal screening. The gene has been mapped to be within a 3.4 Kb segment of the bacterial genome. The intracellular level of the enzyme in strains harboring extrachromosomal copies of topA gene increases with increasing copy number of the gene and the introduction of extrachromosomal copies of the topA gene truncated at its 3' side into a topA strain of E. coli does not significantly influence the expression of the chromosomal copy of topA. These results suggest that the expression of topA is not tightly regulated. Strains in which DNA topoisomerase I is overproduced grow significantly slower in broth and give smaller size colonies on agar plates. Physical mapping of a 20 Kb region containing cysB; topA and trp has also been carried out with a number of restriction enzymes; topA is found to be immediately adjacent to cysB and is separated from trp by a 7 Kb segment where no known gene resides.

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The docking protein p130Cas (Cas) becomes tyrosine-phosphorylated in its central substrate domain in response to extracellular stimuli such as integrin-mediated cell adhesion, and transmits signals through interactions with various intracellular signaling molecules such as the adaptor protein Crk. Src-family kinases (SFKs) bind a specific site in the carboxyl-terminal region of Cas and subsequently SFKs phosphorylate progressively the substrate domain in Cas. In this study crystallography, mutagenesis and binding assays were used to understand the molecular basis for Cas interactions with SFKs. Tyrosine phosphorylation regulates binding of Cas to SFKs, and the primary site for this phosphorylation, Y762, has been proposed. A phosphorylated peptide corresponding to Cas residues 759MEDpYDYVHL767 containing the key phosphotyrosine was crystallized in complex with the SH3-SH2 domain of the SFK Lck. The results provide the first structural data for this protein-protein interaction. The motif in Cas 762pYDYV binds to the SH2 domain in a mode that mimics high-affinity ligands, involving dual contacts of Y762 and V765 with conserved residues in SFK SH2 domains. In addition, Y764 is in position to make an electrostatic contact after phosphorylation with a conserved SFK arginine that mediates interactions with other high-affinity SH2 binders. These new molecular data suggest that Cas may regulate activity of Src as a competing ligand to displace intramolecular interactions that occur in SFKs (between the C-terminal tail and the SH2 domain) and restrain and down-regulate the kinase in an inactive form.