Molecular characterization of protein translocation pores
by Mausumi Ghosh
Date of Examination:2023-12-14
Date of issue:2024-02-27
Advisor:Prof. Dr. Michael Meinecke
Referee:Prof. Dr. Claudia Steinem
Referee:Dr. Alexander Stein
Persistent Address:
http://dx.doi.org/10.53846/goediss-10355
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Abstract
English
Within the intricate realm of intracellular protein transport, individual organelles are reliant upon precise and dedicated protein machineries, commonly referred to as translocon, to facilitate the translocation of proteins to their destined subcellular compartments. In this thesis, we delve into the detailed examination of two unique translocation machineries: peroxisomal protein import mediated by Peroxin (PEX) proteins and the retrotranslocation of misfolded substrates through the Endoplasmic Reticulum-Associated Degradation (ERAD) pathway, in two distinct chapters. Both pathways share the fundamental characteristic of mediating the transport of folded proteins from the cytosol to the peroxisomal matrix or from the ER lumen to the cytosol, respectively. This investigation provides an insightful analysis of the electrophysiological properties inherent to both of these intricate transport mechanisms. Chapter1: Peroxisomes, cellular organelles responsible for lipid metabolism and other critical processes, employ a unique import mechanism that involves peroxisomal translocon, particularly PEX5L. Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and imported post-translationally. Sophisticated protein import systems have developed to facilitate various stages of this process. In humans, PEX5L has been identified as an indispensable component of the peroxisomal translocon. PEX5L serves as the primary receptor for recognizing cargo proteins harbouring a peroxisomal targeting signal (PTS). Cargo proteins bind to the soluble PEX5L in the cytosol, forming a cargo-receptor complex that is subsequently recruited to peroxisomal membranes. At this point, PEX5L interacts with the docking complex PEX13/PEX14, becoming a part of the peroxisomal membrane protein complex that assists in the transfer of cargos into the peroxisomal lumen through a yet unidentified mechanism. Our research reveals that complexes containing PEX5L, purified from human peroxisomal membranes, exhibit characteristics of water-filled pores when reconstituted into planar lipid membranes (PLB). The behaviour of these channels displays high variability in terms of conductance states, selectivity, and voltage and substrate dependent response. Our findings provide evidence of the existence of a PEX5L-associated pore in human peroxisomes, which can be activated by receptor-cargo complexes. Chapter2: The ERAD process is a vital quality control mechanism within the endoplasmic reticulum (ER). It ensures the removal of misfolded proteins from the ER lumen to the cytosol. This process begins with the recognition of misfolded proteins within the ER lumen, followed by their transfer to a pivotal assembly known as the HRD complex. This HRD complex comprises essential components, named Hrd1, Der1, Usa1, and Hrd3. It plays a central role in retrotranslocating misfolded proteins from the ER membrane to the cytosol, where they undergo ubiquitination and subsequent proteasomal degradation. The components involved in molecular mechanisms governing the HRD complex activity and the impact of auto-ubiquitination of the complex on the retrotranslocation process remain subjects of keen interest and investigation. In this thesis, we delve into key facets of HRD complex functionality and explore its electrophysiological characteristics. When the HRD complex is integrated into a lipid bilayer, it exhibits channel activity upon ubiquitination and undergoes partial channel closure upon deubiquitination. We further compare the electrophysiological characteristics of Hrd1 and the HRD complex. We observed a single reversal potential for Hrd1, whereas the HRD complex demonstrated a diverse reversal potential, underscoring the distinct behaviours of its components. Additionally, we observed that gating events in the HRD complex are distinct from those observed in Hrd1. Moreover, our study sheds light on the impact of Hrd1 and Der1 as membrane-thinning factors, causing bilayer destabilization in the planar lipid bilayer (PLB) experiments. This study also hints at the possibility of other components, primarily Hrd1 and Der1, playing a direct role in the retrotranslocation process other than Hrd1 alone.
Keywords: Protein translocation pores
