Project Details
Projekt
Regulation of the peroxisomal protein import pore
Applicants
Dr. Harald Platta; Professorin Dr. Bettina Warscheid
Subject Area
Biochemistry
Term
from 2013 to 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 219314758
Peroxisomal matrix protein import is a highly complex process that relies on the coordi¬nated interaction of numerous proteins to facilitate protein translocation through distinct, transiently formed import pores. Central aim in Project 6 is to reveal the molecular mechanisms involved in the regulation of individual steps of this process with focus on posttranslational modifications. In the first funding period, we found Pex5p and Pex14p, the central components of the import pore for matrix proteins with a peroxisomal targeting signal (PTS) 1, to be highly phosphorylated in vivo. Functional analyses showed that expression of phosphomimetic Pex14p resulted in a partial import defect for PTS1 proteins and that phosphorylation of the PTS1 receptor Pex5p at S232 appears to be a transient event during receptor cycling. We identified several kinases targeting Pex5p and Pex14p at distinct serine and threonine residues and revealed a role for casein kinase 2 in the regulation of the expression of oleic acid-inducible peroxisomal genes essential for pore formation and coding for matrix proteins. Functional analyses of Pex5p ubiquitination, which is known to mark this receptor for recycling (monoubiquitination) or proteasomal degradation (polyubiquitination), allowed for a detailed insight into mechanisms of mono-/polyubiquitination and the ubiquitin-dependent regulation of matrix protein import. Furthermore, we identified the low-molecular mass protein Dyn2p as essential component of a Pex14p/Pex17p/Dyn2p core complex recruited by Pex17p. Establishment and improvement of chemical cross-linking combined with high-resolution mass spectrometry (XL-MS) allowed for identifying intermolecular contact sites in the native Pex14p/Pex17p/Dyn2p complex and site-specific photo-XL-MS was successfully used to identify contact sites and binding steps in a Pex5p-cargo complex. In the new funding period, we will continue with a systematic analysis of the functional significance of individual phosphosites in Pex14p and Pex5p for the regulation of peroxisomal matrix protein import. We will extend this study to essential components of the PTS2 pathway in yeast and to human PEX14 and PEX5. For phosphosites that exhibit a regulatory role, we will identify the kinase mediating the phosphorylation. This will allow us to place the phosphorylation event into the context of cellular signaling networks. We will further analyze crosstalk between ubiquitination and phosphorylation in the N-terminal region of key components of the PTS1 and PTS2 import pore and its significance for peroxisomal matrix protein import. To gain structural information about protein import (sub)complexes and to reveal regulatory mechanisms depending on posttranslational protein modifications, we will extend our XL-MS approach by implementing innovative native MS and comparative XL-MS methodologies.
DFG Programme
Research Units
