Golgi protein homeostasis: Ubiquitination and p97-mediated degradation

• Applicant: Professorin Doris Hellerschmied, Ph.D.
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 577589279

The Golgi apparatus (Golgi) in mammalian cells is the main protein modification and sorting center for secretory and transmembrane proteins and a major signalling hub. Golgi fragmentation and impaired Golgi protein homeostasis are recognized as a hallmark of neurodegenerative diseases and cancer. In cancer cells, Golgi fragmentation can impair protein glycosylation and protein transport. This leads to a change in the proteome of the extracellular matrix, which in turn promotes cancer progression and metastasis. A major limitation in systematically exploring the therapeutic potential of Golgi protein homeostasis is the lack of molecular targets. The protein homeostasis machinery operating at the Golgi, along with related pathways, remains ill-defined. In this proposal, we want to unravel molecular Golgi protein homeostasis pathways guided by candidates identified in a CRISPR/Cas9 knock-out screen challenging proteasomal protein degradation from the Golgi. The post-translational modification of proteins with ubiquitin has recently emerged as a key regulator of Golgi protein homeostasis. We therefore want to understand how the RING-type transmembrane ubiquitin E3 ligases, RNF150 and RNF182, operate at the Golgi. We will define their protein network and their substrate spectrum in pull-down and state-of-the-art proximity labelling studies. In combination with identifying Golgi-related loss-of-function phenotypes, we will map RNF150- and RNF182-driven ubiquitination pathways contributing to Golgi protein homeostasis. Downstream of the ubiquitin signal, the p97 unfoldase facilitates the degradation of Golgi proteins, thereby removing misfolded or damaged proteins and aiding in regulatory proteolysis. We identified two p97 co-factors in our screen – the deubiquitinating enzyme VCPIP1 and UBXD1. VCPIP1 has a proposed role in maintaining Golgi integrity, however its molecular role remains unclear. We have further identified an interaction of the membrane-associated, ubiquitin-binding p97 co-factor FAF2 and a Golgi model substrate. We will explore a direct involvement of FAF2, VCPIP1, and UBXD1 in p97-mediated substrate unfolding and/or degradation at the Golgi. In addition, we will assess Golgi integrity, protein transport capacity and the cellular response to small molecule-induced Golgi stress in VCPIP1 and UBXD1 knock-out cell lines to specify their impact on Golgi homeostasis. The anticipated outcome of this project should reveal how substrate ubiquitination by RNF150 and RNF182 and substrate processing by FAF2, VCPIP1, and UBXD1 together with p97 regulate Golgi protein homeostasis.

• DFG Programme: Research Grants