Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

Abstract

The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97-p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135 function requires membrane association and p97 interaction, both of which are inhibited in mitosis by VCIP135 phosphorylation. We found that wild-type VCIP135, but not its phosphomimetic mutants, rescues Golgi structure in VCIP135-depleted cells. Our results demonstrate that VCIP135 phosphorylation regulates its Golgi membrane association and p97 interaction, and thus contributes to the tight control of the Golgi disassembly and reassembly process during the cell cycle.

Termination of DNA replication forks: “Breaking up is hard to do”

“…The association of p97 with DUBs ultimately lead to de-ubiquitylation of the substrate and prevent its proteasomal degradation (79). Several DUBs have been found to interact with p97 including VCIP135 -a regulator of the p97-mediated Golgi and ER membrane fusion (80,81), YOD1 (and its yeast ortholog Otu1), USP13, ataxin3 and USP25 -linked to the regulation of ERAD (79,(82)(83)(84)(85) and USP50 -which has been implicated in the regulation of the DNA damage checkpoint (84,86).…”

Monoubiquitination of Syntaxin 5 Regulates Golgi Membrane Dynamics during the Cell Cycle

“…3B). To investigate whether ubiquitinated Syn5 is a direct substrate of VCIP135, in vitro ubiquitinated Syn5 was treated with recombinant VCIP135 (Zhang and Wang, 2015; Zhang et al, 2014). As shown in Fig.…”

“…It has been shown that the DUB activity of VCIP 135 is inactivated by phosphorylation in mitosis and reactivated by dephosphorylation in late mitosis (Zhang and Wang, 2015; Zhang et al, 2014). To determine the temporal dynamics of VCIP135-mediated Syn5 deubiquitination during cell cycle progression, HeLa cells were arrested in early mitosis by nocodazole treatment, released by washing out the drug to allow cells progress to later mitosis and G1 phase (Zhang and Wang, 2015), and followed by immunoprecipitation of endogenous VCIP135 at different time points.…”

“…Ubiquitination is mediated by the Golgi-localized ubiquitin E3 ligase HACE1 (Tang et al, 2011). Deubiquitination requires the deubiquitinase VCIP135 (Wang et al, 2004), whose activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and re-activated upon dephosphorylation in telophase (Zhang and Wang, 2015; Zhang et al, 2014). The ubiquitination substrate, however, has remained unknown for many years.…”

“…Prior to membrane fusion in telophase, ubiquitin is removed by the deubiquitinating (DUB) enzyme VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135) (Wang et al, 2004). VCIP135 activity is regulated by phosphorylation in the cell cycle; it is inactivated by phosphorylation in metaphase and reactivated upon dephosphorylation in telophase (Zhang and Wang, 2015; Zhang et al, 2014). In this study, we report the identification of Syn5 as the ubiquitinated substrate on the Golgi membrane and the mechanism of ubiquitination in Golgi disassembly and reassembly during the cell cycle.…”