In the ubiquitin-proteasome system, a subset of ubiquitylated proteins requires the AAA+ ATPase p97 (also known as VCP or Cdc48) for extraction from membranes or protein complexes before delivery to the proteasome for degradation. Diverse ubiquitin adapters are known to link p97 to its client proteins, but two recent papers on the adapter protein UBXD7, including one by Bandau et al. in BMC Biology, suggest that rather than simply linking p97 to ubiquitylated proteins, this adapter may be essential to coordinate ubiquitylation and p97-mediated extraction of the proteasome substrate. These findings add to growing indications of richly diverse roles of adapters in p97-mediated signaling functions.

The ubiquitin-proteasome system mediates degradation of misfolded or damaged proteins to ensure cellular protein homeostasis, and selectively removes regulatory proteins in critical signaling pathways. The key step is the posttranslational modification of the substrate with the small protein ubiquitin and extension to ubiquitin chains that serve as a signal for degradation by the proteasome. The ubiquitylation reaction is mediated by a cascade of enzymes, comprising the E1 ubiquitin-activating enzyme, an E2 conjugating enzyme and an E3 ligase that functions in the attachment of the ubiquitin to the substrate (Figure ​(Figure1).1). Ubiquitin-binding shuttling factors then deliver the ubiquitylated substrates to the proteasome. The conserved hexameric AAA+ ATPase p97 has emerged as an important player during this step for a subset of client proteins [1,2]. Unlike other shuttle proteins, which simply bind both to ubiquitin chains and to components of the proteasome, p97 uses the energy of ATP hydrolysis to structurally remodel or unfold its clients, and is believed thus to help extract them from cellular structures, segregate them from binding partners or generate initial unfolded stretches to facilitate degradation by the proteasome. This is required for degradation of proteins associated with the endoplasmic reticulum, with the outer mitochondrial membrane, and with chromatin, and for some soluble proteins [1,2]. The interaction of the p97 ATPase with the ubiquitylated substrate is mediated by diverse ubiquitin adapters [1,2], which recognize both p97 and the ubiquitin chain on its client protein. Members of the largest family of such adapters are characterized by UBX and UBX-like domains, which assume a ubiquitin-fold and bind the amino-terminal domain of p97 [3]. They also contain ubiquitin binding domains (UBDs), including the UBA domain, that recognize the client. The domain structures of p97 and its UBD-UBX adapters are schematically illustrated in Figure ​Figure2.2. In a simple linear view, p97-adapter complexes bind substrates after ubiquitylation and then deliver them to the proteasome (Figure ​(Figure11).

The starting point for the recent investigations was a mass spectrometry study by Alexandru and colleagues in Raymond Deshaies' group that revealed a far-reaching interaction network between p97 and a large number of E3 ligases, including cullin RING ligases (CRLs) [4]. Although for simplicity the E3 ligase in Figure ​Figure11 is schematically shown as a single entity, many E3 ligases are in fact complexes of several subunits with distinct functions. CRLs are among these multisubunit complexes, and consist of a cullin scaffold (CUL1, 2, 3, 4A, 4B or 5), a RING-domain protein that recruits an E2, and, except for CUL3-based CRLs, a cullin-specific adapter that in turn binds one of many substrate recruitment factors. In cullin-RING E3 ubiquitin ligases, the ubiquitin is directly transferred from the E2 enzyme to the substrate, the role of the cullin-RING being to recruit E2 and target protein and position them appropriately for ubiquitin transfer. Taking into account the different possible combinations of subunits, CRLs constitute up to 240 different ligase complexes that greatly extend the potential substrate spectrum of p97. Alexandru et al. [4] established the functional relevance of p97 binding to CRLs by showing that the degradation of the hypoxia-inducible factor HIF1α, a CUL2 substrate that is constitutively degraded in normoxic conditions, depends upon p97. Indeed, other CRL substrates have in the meantime been shown to require p97 for extraction: these include the mitotic kinase Aurora B and the polymerase II catalytic subunit Rpb1, which are ubiquitylated by CUL3, as well as the replication licensing factor Cdt1, which is targeted by CUL4A [1].

What might be the functional significance of UBXD7 binding to neddylated CRLs? In the case of the Cul3 substrate Rpb1 in yeast, the UBXD7 orthologue Ubx5 (and specifically its UIM) is required together with p97 for efficient degradation [6]. This suggests that linking substrate ubiquitylation and p97-mediated extraction may be important for efficient degradation of some substrates. Surprisingly, however, in the case of the CUL2 substrate HIF1α, p97 is required, but UBXD7 is not. On the contrary, Alexandru and colleagues found that UBXD7 depletion in cells accelerates HIF1α degradation [5]. Consistent with this, overexpression of wild-type UBXD7 inhibits HIF1α ubiquitylation, an effect that can be shown to be dependent on the presence of the UIM [5]. These observations suggest that UBXD7 acts as an antagonist of substrate ubiquitylation and degradation. This would not be surprising given the pivotal role of the UBXD7 docking site on CRL in the mechanism of CRL activation by neddylation, which involves NEDD8 and adjacent regions. NEDD8 modification induces conformational changes in the CRL that position the E2 in the vicinity of the substrate and thus promote ubiquitin transfer [7]. Indeed, Deshaies and colleagues mention that UBXD7 binding interferes with recruitment of the E2 Cdc34 to the CUL1 SCF^β-TrCP ligase and reduces substrate ubiquitylation [6]. Moreover, proper CRL activity requires NEDD8 to cycle on and off the cullin, while UBXD7 binding locks the CRL in the neddylated form. The function of a factor affecting such delicately balanced properties may be difficult to interpret because effects may be diverse depending on the experimental conditions.

The new insights into UBXD7 function add to accumulating evidence that p97-adapter complexes are more multifaceted and their function is more complex than in a simple linear model, where the adapters' sole role is to link the ubiquitylated client to p97 ATPase (Figure ​(Figure3).3). One indicator of this complexity is that p97 can associate with several cofactors at a time. An emerging model suggests that a subset of cofactors including Ufd1-Npl4 or p47 constitute major adapters that can define core complexes and may even govern association of additional cofactors specific for particular pathways. One class of additional cofactors comprises targeting factors that recruit p97 to a specific subcellular location. A good example is the membrane protein UBXD8 (also called FAF2, or Ubx2 in yeast) that recruits p97-Ufd1-Npl4 to function in endoplasmic reticulum-associated degradation [2].