Sehr, J. | Search bioRxiv

bioRxiv2022

Peroxisomal ATPase ATAD1 acts in quality control of the protein import machinery

ATAD1 is an AAA-ATPase which shows a dual localization at mitochondria and peroxisomes. While its peroxisomal function is not known, in mitochondria ATAD1 is part of a quality control mechanism extracting mislocalised tail-anchored and accumulated precursor proteins from the outer membrane. Here, we studied the peroxisomal interactome of ATAD1 and could show that human ATAD1 interacts with PEX5, a cytosolic receptor for peroxisomal matrix proteins which transiently inserts into peroxisomal membranes. Upon cargo-release, mono-ubiquitinated PEX5 is recycled into the cytosol by the AAA-peroxins PEX1 and PEX6. The accumulation of ubiquitinated PEX5 is known to trigger degradation of whole organelles called pexophagy. Here, we used ATAD1-, PEX1- and ATAD1/PEX1-CRISPR-Knockout cell lines to investigate the physiological role of an ATAD1-PEX5 interaction. We could show an influence of ATAD1 on the stability of accumulated PEX5 and hypothesize a role in a peroxisomal quality control mechanism enabling clearance of ubiquitinated receptor from the membrane.

biochemistry↗

bioRxiv2021

Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5

Measuring diffusion dynamics in living cells is essential for the understanding of molecular interactions. While various techniques have been used to explore such characteristics in the plasma membrane, this is less developed for measurements inside the cytosol. An example of cytosolic action is the import of proteins into peroxisomes, via the peroxisomal import receptor PEX5. Here, we combined advanced microscopy and spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and super-resolution STED microscopy to present a detailed characterization of the diffusion and interaction dynamics of PEX5. Among other features, we disclose a slow diffusion of PEX5, independent of aggregation or target binding, but associated with cytosolic interaction partners via its N-terminal domain. This sheds new light on the functionality of the receptor in the cytosol. Besides specific insights, our study highlights the potential of using complementary microscopy tools to decipher molecular interactions in the cytosol via studying their diffusion dynamics. SummaryThe peroxisomal import receptor PEX5 transports newly synthesized proteins from the cytosol to the peroxisomal matrix. Here the cytosolic diffusion and interaction dynamics of PEX5 are characterized by advanced microscopic spectroscopy methods, revealing a so far unknown interaction partner.

biophysics↗