Endolysosomal sorting of ubiquitylated caveolin-1 is regulated by VCP and UBXD1 and impaired by VCP disease mutations

Abstract

The AAA-ATPase VCP/p97 cooperates with distinct cofactors to process ubiquitinated proteins in different cellular pathways 1–3. VCP missense mutations cause a systemic degenerative disease in humans, but the molecular pathogenesis is unclear 4, 5. We used an unbiased mass spectrometry approach and identified a VCP complex with the UBXD1 cofactor, which binds the plasma membrane protein caveolin-1 (Cav1) and whose formation is specifically disrupted by disease-associated mutations. We show that VCP-UBXD1 targets mono-ubiquitinated Cav1 in SDS-resistant high molecular weight complexes on endosomes, which are en route to degradation in endolysosomes 6. Expression of VCP mutant proteins, chemical inhibition of VCP, or siRNA-mediated depletion of UBXD1 leads to a block of Cav1 transport at the limiting membrane of enlarged endosomes in cultured cells. In patient muscle, muscle-specific Caveolin-3 (Cav3) accumulates in sarcoplasmic pools and specifically delocalises from the sarcolemma. These results extend the cellular functions of VCP to mediating sorting of ubiquitinated cargo in the endocytic pathway and suggest that impaired trafficking of caveolin may contribute to the pathogenesis in individuals with VCP mutations.

Control of p97 function by cofactor binding

“…Specifically, increased amounts of p47, UFD1-NPL4, and ataxin-3, but decreased amounts of UBXD1 and UBE4B have been found. Interestingly, the decreased binding to UBXD1 is associated with a blockage of the endolysosomal trafficking of caveolin 1 [10]. Although disease mutants and wild-type p97 interact with both cofactors in a similar manner in vitro, disease mutants no longer can be activated by p37, but are still inhibited by p47 [112].…”

“…Due to an altered inter-subunit communication in IBMPFD mutants the D1 domains fail to regulate their respective nucleotide-binding states as evidenced by a lower amount of pre-bound ADP and weaker affinity for ADP, resulting in a better accessibility for ATP in comparison to the wild-type protein [29,30]. The altered conformational equilibrium of the N domain in p97 disease mutants is accompanied by elevated ATPase activities in vitro [43,115,116] and altered interactions with some but not all cofactors in cells, which has been proposed to be key to the pathogenesis of IBMPFD [10,[116][117][118] (for a recent review see [119]). Specifically, increased amounts of p47, UFD1-NPL4, and ataxin-3, but decreased amounts of UBXD1 and UBE4B have been found.…”

“…In higher eukaryotes, the p97 UBXD1 complex represents a third, distinct p97 assembly with so far poorly defined function(s) in endolysosomal trafficking [10]. Even though UBXD1 belongs to the UBX family of p97 cofactors, it does not bind p97 via its UBX domain because the R. .…”

“…A subsequent bioinformatic search defined a minimal VIM consensus sequence and identified several additional VIM-containing proteins [63]. These include ANKZF1 (ANKyrin repeat and Zinc Finger domain-containing-1, also known as ZNF744 or Vms1), a protein that has been implicated in mitochondrial protein degradation [64]; the UBX protein UBXD1, involved in sorting of ubiquitylated cargo in the endocytic pathway [10]; AIRAPL (Arsenite Inducible RNA Associated Protein Like Protein, also known as ZFAND2B), an ER membrane-anchored protein which associates with and regulates the function of the 26S proteasome under arsenite stress [65]; SelS (also called VIMP, VCP-Interacting Membrane Protein), a protein involved in the recruitment of p97 to the ER membrane [66]; and the yeast protein Wss1, a metalloprotease involved in DNA-protein crosslink repair [67]. With the exception of SelS it was shown that these proteins interact via their VIM with p97 [63,64,[67][68][69].…”

“…On the cellular level, both diseases are characterized by cytoplasmic protein inclusions (aggregates) positive for ubiquitin and the Tar DNA-binding Protein 43 . Analysis of IBMPFD patient-derived cell-lines and IBMPFD mouse models revealed that disease-causing point mutations in VCP affect autophagy and endolysosomal protein degradation [9,10].…”