Abstract
The protein known as p97 or VCP in mammals and Cdc48 in yeast is a versatile ATPase complex involved in several biological functions including membrane fusion, protein folding, and activation of membrane-bound transcription factors. In addition, p97 plays a central role in degradation of misfolded secretory proteins via the ER-associated degradation pathway. This functional diversity of p97 depends on its association with various cofactors, and to further our understanding of p97 function it is important that these cofactors are identified and analyzed. Here, we isolate and characterize the human protein named Rep8 or Ubxd6 as a new cofactor of p97. Mouse Rep8 is highly tissue-specific and abundant in gonads. In testes, Rep8 is expressed in post-meiotic round spermatids, whereas in ovaries Rep8 is expressed in granulosa cells. Rep8 associates directly with p97 via its UBX domain. We show that Rep8 is a transmembrane protein that localizes to the ER membrane with its UBX domain facing the cytoplasm. Knock-down of Rep8 expression in human cells leads to a decreased association of p97 with the ER membrane and concomitantly a retarded degradation of misfolded ER-derived proteasome substrates. Thus, Rep8 tethers p97 to the ER membrane for efficient ER-associated degradation.
Highlights
When correct folding of proteins or assembly of oligomeric proteins in the endoplasmic reticulum (ER) is disturbed, misfolded or unassembled proteins accumulate inside the ER lumen
The UBX domain is regarded as a general p97-interacting domain [24], indicating that Rep8 was a valid target of p97 in the yeast two-hybrid screen
Rep8 associates with Hrd1 Since we found that Rep8 is a cofactor of p97 that localizes to the ER membrane, we analyzed if Rep8 was associated with the E3 ubiquitin-protein ligase, Hrd1, that plays an important role in ERassociated degradation (ERAD) [8]
Summary
When correct folding of proteins or assembly of oligomeric proteins in the endoplasmic reticulum (ER) is disturbed, misfolded or unassembled proteins accumulate inside the ER lumen If such proteins are allowed to linger, they may form insoluble aggregates and pose a serious threat to the cell. The ATP-powered conformational changes allow p97 to drive the disassembly of protein complexes and segregate proteins from their binding partners [16,17] This catalytic activity of p97, termed the ‘‘segregase’’ activity [10,18], is probably restricted to ubiquitylated proteins and is important for a number of cellular pathways, including membrane fusion [19], protein degradation [11,20], and transcription factor maturation [18,21]. Ubxd1 [25,26], all characterized UBX-domain proteins associate directly with p97 via their UBX domains [24,27,28]
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