We have reconstituted the ubiquitination pathway for the Cdk inhibitor Sic1 using recombinant proteins. Skp1, Cdc53, and the F-box protein Cdc4 form a complex, SCFCdc4, which functions as a Sic1 ubiquitin-ligase (E3) in combination with the ubiquitin conjugating enzyme (E2) Cdc34 and E1. Cdc4 assembled with Skp1 functions as the receptor that selectively binds phosphorylated Sic1. Grr1, an F-box protein involved in Cln destruction, forms complexes with Skp1 and Cdc53 and binds phosphorylated Cln1 and Cln2, but not Sic1. Because the constituents of the SCF complex are members of protein families, SCFCdc4 is likely to serve as the prototype for a large class of E3s formed by combinatorial interactions of related family members. SCF complexes couple protein kinase signaling pathways to the control of protein abundance.

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinases (CDKs) by an unknown mechanism. We show that the Cdc14 phosphatase triggers mitotic exit by three parallel mechanisms, each of which inhibits Cdk activity. Cdc14 dephosphorylates Sic1, a Cdk inhibitor, and Swi5, a transcription factor for SIC1, and induces degradation of mitotic cyclins, likely by dephosphorylating the activator of mitotic cyclin degradation, Cdh1/Hct1. Feedback between these pathways may lead to precipitous collapse of mitotic CDK activity and help coordinate exit from mitosis.

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.

In budding yeast, ubiquitination of the cyclin-dependent kinase (Cdk) inhibitor Sic1 is catalyzed by the E2 ubiquitin conjugating enzyme Cdc34 in conjunction with an E3 ubiquitin ligase complex composed of Skp1, Cdc53 and the F-box protein, Cdc4 (the SCF Cdc4 complex). Skp1 binds a motif called the F-box and in turn F-box proteins appear to recruit specific substrates for ubiquitination. We find that Skp1 interacts with Cdc53 in vivo, and that Skp1 bridges Cdc53 to three different F-box proteins, Cdc4, Met30, and Grr1. Cdc53 contains independent binding sites for Cdc34 and Skp1 suggesting it functions as a scaffold protein within an E2/E3 core complex. F-box proteins show remarkable functional specificity in vivo: Cdc4 is specific for degradation of Sic1, Grr1 is specific for degradation of the G 1 cyclin Cln2, and Met30 is specific for repression of methionine biosynthesis genes. In contrast, the Cdc34-Cdc53-Skp1 E2/E3 core complex is required for all three functions. Combinatorial control of SCF complexes may provide a basis for the regulation of diverse cellular processes.

The ubiquitin system of intracellular protein degradation controls the abundance of many critical regulatory proteins. Specificity in the ubiquitin system is determined largely at the level of substrate recognition, a step that is mediated by E3 ubiquitin ligases. Analysis of the mechanisms of phosphorylation directed proteolysis in cell cycle regulation has uncovered a new class of E3 ubiquitin ligases called SCF complexes, which are composed of the subunits Skp1, Rbx1, Cdc53 and any one of a large number of different F-box proteins. The substrate specificity of SCF complexes is determined by the interchangeable F-box protein subunit, which recruits a specific set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34. F-box proteins have a bipartite structure--the shared F-box motif links F-box proteins to Skp1 and the core complex, whereas divergent protein-protein interaction motifs selectively bind their cognate substrates. To date all known SCF substrates are recognised in a strictly phosphorylation dependent manner, thus linking intracellular signalling networks to the ubiquitin system. The plethora of different F-box proteins in databases suggests that many pathways will be governed by SCF-dependent proteolysis. Indeed, genetic analysis has uncovered roles for F-box proteins in a variety of signalling pathways, ranging from nutrient sensing in yeast to conserved developmental pathways in plants and animals. Moreover, structural analysis has revealed ancestral relationships between SCF complexes and two other E3 ubiquitin ligases, suggesting that the combinatorial use of substrate specific adaptor proteins has evolved to allow the regulation of many cellular processes. Here, we review the known signalling pathways that are regulated by SCF complexes and highlight current issues in phosphorylation dependent protein degradation.

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses.

Simian foamy viruses (SFVs) are ubiquitous in non-human primates (NHPs). As in all retroviruses, reverse transcription of SFV leads to recombination and mutation. Because more humans have been shown to be infected with SFV than with any other simian borne virus, SFV is a potentially powerful model for studying the virology and epidemiology of viruses at the human/NHP interface. In Asia, SFV is likely transmitted to humans through macaque bites and scratches that occur in the context of everyday life. We analyzed multiple proviral sequences from the SFV gag gene from both humans and macaques in order to characterize retroviral transmission at the human/NHP interface in Bangladesh. Here we report evidence that humans can be concurrently infected with multiple SFV strains, with some individuals infected by both an autochthonous SFV strain as well as a strain similar to SFV found in macaques from another geographic area. These data, combined with previous results, suggest that both human-facilitated movement of macaques leading to the introduction of non-resident strains of SFV and retroviral recombination in macaques contribute to SFV diversity among humans in Bangladesh.

e Foamy viruses (FV) are complex retroviruses that naturally infect all nonhuman primates (NHP) studied to date. Zoonotic transmission of Old World NHP simian foamy viruses (SFV) has been documented, leading to nonpathogenic persistent infections. To date, there have been no reports concerning zoonotic transmission of New World monkey (NWM) SFV to humans and resulting infection. In this study, we developed a Western blot assay to detect antibodies to NWM SFV, a nested PCR assay to detect NWM SFV DNA, and a ␤-galactosidase-containing indicator cell line to assay replication of NWM SFV. Using these tools, we analyzed the plasma and blood of 116 primatologists, of whom 69 had reported exposures to NWM. While 8 of the primatologists tested were seropositive for SFV from a NWM, the spider monkey, none had detectable levels of viral DNA in their blood. We found that SFV isolated from three different species of NWM replicated in some, but not all, human cell lines. From our data, we conclude that while humans exposed to NWM SFV produce antibodies, there is no evidence for long-term viral persistence. Foamy viruses (FV) are unusual complex retroviruses that infect cattle, horses, cats, and all species of nonhuman primates (NHP) examined to date (reviewed in reference 1). Simian foamy viruses (SFV) can cause life-long infections in natural hosts without any apparent pathogenicity (2). In cell culture models, SFV can establish latent infections in some cell types and lytic infections in others, resulting in cytopathic effects (CPE) that include syncytium formation (reviewed in reference 3). In infected macaques, FV DNA is found in almost all tissues, while FV RNA and replicating virus are limited to the superficial epithelial cells of the oral mucosa in immunocompetent animals (4, 5). Consistent with the site of viral replication detected in vivo, it is thought that FV are transmitted via saliva, through grooming, biting, and other behaviors. Studies of natural transmission suggest that infant and young NHP are resistant to infection, presumably because of passive immunity from maternal antibodies, but typically (for macaques) become infected by 3 years of age (6). Epidemiological studies indicate that SFV seroprevalence can reach up to 100% in adults (reviewed in reference 7).Although FV do not naturally infect humans, SFV have been found to be zoonotically transmitted, most likely through contact with saliva from an infected NHP. Several studies have documented the transmission of SFV to humans who interact directly with Old World (OW) NHP, including Cercopithicus species, baboons, macaques, mandrills, gorillas, and chimpanzees (reviewed in reference 7). SFV antibody-positive humans have been found in a variety of natural settings, including people in Asia who live in areas with free-ranging macaques, villagers in Gabon with known exposure to NHP, and a population of hunters in Cameroon with bites from Old World NHP (6,(8)(9)(10)(11). SFV antibody-positive humans have also been documented in various laboratory, veterina...