Lessons from Fungal F-Box Proteins

The F-box domain, so called after a conserved domain found in human cyclin F (5), was described in 1996 (6) after first being denoted a conserved N-terminal domain found in a subset of proteins (110). The F-box hypothesis was introduced shortly after (162, 185) and holds that F-box-containing proteins (henceforth F-box proteins) act as scavengers in the cell, collecting “junk” proteins to deliver to a “waste processor,” called the SCF complex, to which they dock through their F-box domain. In the SCF complex, the junk proteins are marked with ubiquitin for “incineration” in the proteasome. F-box proteins do not act indiscriminately but recruit specific, often modified proteins to the SCF complex and in this way regulate the level of certain proteins in a cell. F-box proteins are found in all eukaryotes and display a large variety of functions. In fungi they are, for example, involved in control of the cell division cycle, glucose sensing, mitochondrial connectivity, and control of the circadian clock. F-box proteins are commonly identified by the presence of a stretch of primary sequence that matches the consensus for an F-box domain (Fig. ​(Fig.1).1). However, it can be questioned whether just the occurrence of an F-box domain in a protein sequence is sufficient to assume compliance with the F-box hypothesis. The F-box hypothesis is based on the assumption that an F box mediates assembly into an SCF complex through binding to the Skp1 subunit (Fig. ​(Fig.2).2). The SCF complex consists of Skp1 (suppressor of kinetochore protein mutant) (34), Cul1 (Cullin) (135), Rbx1 (ring-box protein) (86), and an F-box protein and catalyzes (like other E3 ligases), in cooperation with the E1 and E2 enzymes, the transfer of the small protein ubiquitin to the target protein (108, 162). Among fungi, the methods of regulation of the SCF complexes and hence the methods of regulation of the F-box proteins in these complexes appear to differ. SCF complexes are likely activated and regulated through a recycling mechanism (35), which involves three main contributors: the neddylator protein DCN1, responsible for the transfer of Nedd8 to Cul1 (112, 133, 155, 159, 174, 211); the deneddylator CSN (COP9 signalosome) (136, 137, 146, 179, 200) (reviewed in references 159, 201, and 208); and the CAND1 protein (127, 215), which binds to deneddylated Cul1 and competes out the Skp1-F-box complex from the core of the SCF complex. A new round of neddylation removes CAND1 and thereby creates binding space for a new Skp1-F-box complex. In budding yeast (Saccharomyces cerevisiae), deletion mutants for Nedd8 and CSN5, the CSN subunit responsible for the deneddylation reaction, are both viable (36, 115, 124). This means that, although the components of the SCF recycling mechanism are present, this process is not required for survival. A second difference in budding yeast in comparison to other fungi is that is does not have the CAND1 protein, adding to the notion that in budding yeast recycling acts differently. In fission yeast (Schizosaccharomyces pombe), CAND1 is present, and Nedd8 is required for survival (155), but the CSN5 subunit is not (145, 216). Apparently, in fission yeast, the neddylation reaction is required for proper SCF function, but deneddylation is not, suggesting that in fission yeast an alternative deneddylation may be present. In Neurospora crassa, a deletion mutant for subunit 2 of CSN, Δcsn-2, is viable but lacks a normal circadian rhythm and conidiation (64), while in Aspergillus nidulans, four CSN mutants, including one for subunit 5 (Δcsne), all lack fruiting body development (25, 26). Together, these data suggest that, in filamentous fungi, proper recycling of the SCF is strictly required only for certain developmental processes, in accordance with the requirements of CSN in development in more-complex, multicellular organisms (reviewed in reference 179). FIG. 1. F-box consensus sequence. The motif is about 45 amino acids long and based on the HMM logo for the F-box motif (178). Highly conserved amino acids are underlined, and the two most conserved amino residues, the leucine and proline at positions 6 and 7, ... FIG. 2. SCF complex and ubiquitination of target proteins. The SCF complex functions within the ubiquitination reaction through combined action with the E1 and E2 enzymes. F-box proteins bind to Skp1 via their F-box domain and to targets via their C-terminal ... Some F-box proteins appear to function without binding to Skp1, suggesting that not all F-box proteins take part in an SCF complex. This also means that not all proteins interacting with an F-box protein will be ubiquitinated and proteasomally degraded. In another deviation from the F-box hypothesis, some F-box protein/Skp1 complexes do not seem to be involved in ubiquitination. Furthermore, even when an F-box domain mediates assembly into an SCF complex, the result may be self-ubiquitination rather than fulfillment of a scavenger function. Since 1996, several review articles covering the emerging theme of ubiquitin-mediated protein degradation and the widespread occurrence of F-box proteins have been published (38, 68, 71, 72, 92, 118, 198, 204, 205). Here, we discuss fungal F-box proteins, including their targets (if identified), and when possible, classify these F-box proteins according to degree of compliance with the F-box hypothesis. Most literature on fungal F-box proteins covers those found in budding yeast and, to a lesser extent, fission yeast, but important findings have also been reported for filamentous ascomycetes. In Table ​Table1,1, fungal F-box proteins described in the literature are listed according to their main cellular function. The distantly related budding and fission yeasts share 10 (likely) orthologous F-box proteins. Budding yeast contains an additional 11 F-box proteins and fission yeast 7 (89). Cdc4, Grr1, and Met30 from budding yeast and their counterparts in other fungi are the most studied fungal F-box proteins and are conserved throughout the fungal kingdom. In total, 31 F-box proteins are discussed, exclusively from ascomycetes: the “model” fungi S. cerevisiae, S. pombe, Kluyveromyces lactis, A. nidulans, Hypocrea jecorina, and N. crassa and the pathogenic fungi Candida albicans, Fusarium graminearum, Fusarium oxysporum, and Magnaporthe grisea. TABLE 1. Fungal F-box proteins described in the literature and discussed in this review