Function and molecular mechanism of signal peptide peptidase (SPP) in regulated membrane protein abundance control

• Applicant: Professor Dr. Marius Lemberg
• Subject Area: Biochemistry ; Cell Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407070872

Intramembrane proteases have the unusual property of cleaving peptide bonds within the lipid bilayer of cellular membranes, an environment not obviously suited to a water-requiring hydrolysis reaction. These unusual enzymes include S2P, rhomboids, RCE1, presenilin/γ-secretase and signal peptide peptidase (SPP) and SPP-like proteases. Intramembrane proteolysis was initially identified with its common role in the selective release of bioactive peptides from the membrane, but we hypothesize that these evolutionary widespread proteases are primarily involved in the control of membrane protein homeostasis. As a proof of concept for this idea, we have recently showed that the yeast SPP orthologue Ypf1 regulates protein turnover at the Endoplasmic Reticulum (ER). We have termed this pathway, which controls the levels of nutrient transporters according to cellular needs, ERAD-R (ER Associated Degradation-Regulatory). Consistent with such a function, we and others showed that human SPP functionally interacts with the ERAD factor Derlin1 and the E3 ubiquitin ligase TRC8 forming a 500-kDa complex that cleaves certain tail-anchored and type II membrane proteins: The parallels to Ypf1 suggest an evolutionarily conserved mechanism. However, the molecular function of SPP in ERAD and its physiological role in humans are still ill defined. Our recent unpublished work demonstrates that human SPP controls the abundance of the squalene synthase FDFT1 that serves as a key branching point between cholesterol synthesis and a non-sterol branch of the isoprenoid pathway. Here, we propose a study to systematically define the physiological substrate spectrum of SPP in tissue culture cells by quantitative proteomics and to analyze the molecular mechanism of ERAD-R in regulated protein abundance control. Specific goals are: 1.) Systematic identification of physiological SPP substrates 2.) Studying the function of SPP-mediated ERAD-R 3.) Deciphering regulatory principles and mechanismsTo this end, we aim to study the human SPP in established cell-based assays and to reconstitute the SPP ERAD complex in vitro. Taken together, this project will open a new view on protein homeostasis control in eukaryotic cells, with important implications in a broad variety of essential cellular processes ranging from regulation of lipid biosynthesis to abundance control of polytopic membrane proteins.

• DFG Programme: Research Grants