Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion (''bait'') in a MATa strain and as an activation domain fusion (''prey'') in a MAT␣ strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of Ϸ4 ؋ 10 6 different combinations, constituting Ϸ10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA‐replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c‐mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant‐negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos‐mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.

The c‐mos proto‐oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N‐terminal residue, or by a rule referred to here as the ‘second‐codon rule’. We demonstrate that unstable Mos is degraded by the ubiquitin‐dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N‐terminus of Mos from being recognized by a ubiquitin‐protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N‐terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.

The c‐mos proto‐oncogene product (Mos), an essential component of the cytostatic factor responsible for meiotic arrest in vertebrate eggs, undergoes specific proteolysis soon after fertilization or activation of Xenopus eggs. To determine the degradation pathway of Mos on egg activation, various Mos mutants were expressed in Xenopus eggs and their degradation on egg activation was examined. Mos degradation absolutely required its penultimate proline (Pro2) residue and dephosphorylation of the adjacent serine (Ser3) residue. These degradation signals were essentially the same as those of Mos in meiosis I of Xenopus oocyte maturation, where Mos has been shown to be degraded by the ‘second‐codon rule’‐based ubiquitin pathway. To test whether Mos degradation on egg activation is also mediated by the ubiquitin pathway, we attempted to identify and abrogate a specific ubiquitination site(s) in Mos. We show that the major ubiquitination site in Mos is a Lys34 residue and that replacement of this residue with a non‐ubiquitinatable Arg residue markedly enhances the stability of Mos on egg activation. These results indicate that the degradation of Mos on egg activation or fertilization is mediated primarily by the N‐terminal Pro2‐dependent ubiquitin pathway, as in meiosis I of oocyte maturation. The N‐terminal Pro2 residue of Mos appears to be naturally selected primarily for its degradation on fertilization, rather than that in meiosis I.