Final Report Summary - PEROXISCOPY (Morphogenesis of de novo peroxisome formation)
WP1. Spotting the intermediate stages of de novo peroxisome formation
It was demonstrated that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells, as these cells already contain reticular and vesicular structures, which harbour key proteins of the peroxisomal receptor docking complex, Pex13 and Pex14, as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). The structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible for autophagic degradation, which is blocked in this double mutant. These data indicate, contrary to earlier suggestions, that peroxisomes are not formed de novo from the ER when the PEX3 gene is re-introduced in pex3 cells. Instead, it was found that re-introduced Pex3 sorts to the peroxisomal structures in pex3 cells, after which they mature into normal peroxisomes. These results have been published in the Journal of Cell Biology. (Knoops et al. (2014) Pex3 is not required for the formation of peroxisomal membrane vesicles. J Cell Biol. 2014 Mar 3;204(5):659-68.)
WP2. Correlative light and electron microscopy (CLEM) and electron tomography
Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of peroxisomal membrane remnants that are present in the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high resolution electron microscopy techniques. We observed that these structures lack matrix proteins, but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13 and Pex14. Moreover, we show that upon reintroduction of Pex1 in Pex1-deficient cells, these membrane remnants (ghosts) rapidly (within 2h) incorporate matrix proteins and develop into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import rather than being involved in fusion of endoplasmic reticulum-derived peroxisomal vesicles. The revised manuscript describing these results has been submitted for publication in the Journal of Cell Biology. (Knoops et al., Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1. Submitted)
The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. We identified an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrated that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. In particular using electron tomography, we were able to show that, although it seemed that more peroxisomes were present in a dnm1 deletion strain that overexpressed Pex11p, they are all connected via small neck-like membrane connections. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Our work identifies a previously unknown requirement for a GAP in DLP function. This results have been published in the Proceedings of the National Academy of Sciences. (Williams et al. (2015) The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission. Proc Natl Acad Sci U S A. 2015 May 19;112(20):6377-82.)
WP3. Analysis of in vitro peroxisome formation.
I have visited the laboratory of Prof. Randy Schekman during spring 2013. This visit has been very inspiring and I repeated the experiments that were published before. However, the setup of their experiment, i.e. microsomes isolated from pex19 cells which would generate vesicles upon in vitro addition of Pex19, raises questions about the true mechanism of vesicle formation. Since I recently demonstrated that vesicles are already present in pex19 cells, the main question arises whether the vesicles in the in vitro system are already present during microsome isolation (only associated to the ER e.g. via Inp1 interaction) and thus are not the result of an actual budding event. At this moment we are optimizing conditions for visualization of these vesicles as well as normal mature peroxisomes in a cryo-electron microscopy approach like stated in WP3.
It was demonstrated that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells, as these cells already contain reticular and vesicular structures, which harbour key proteins of the peroxisomal receptor docking complex, Pex13 and Pex14, as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). The structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible for autophagic degradation, which is blocked in this double mutant. These data indicate, contrary to earlier suggestions, that peroxisomes are not formed de novo from the ER when the PEX3 gene is re-introduced in pex3 cells. Instead, it was found that re-introduced Pex3 sorts to the peroxisomal structures in pex3 cells, after which they mature into normal peroxisomes. These results have been published in the Journal of Cell Biology. (Knoops et al. (2014) Pex3 is not required for the formation of peroxisomal membrane vesicles. J Cell Biol. 2014 Mar 3;204(5):659-68.)
WP2. Correlative light and electron microscopy (CLEM) and electron tomography
Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of peroxisomal membrane remnants that are present in the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high resolution electron microscopy techniques. We observed that these structures lack matrix proteins, but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13 and Pex14. Moreover, we show that upon reintroduction of Pex1 in Pex1-deficient cells, these membrane remnants (ghosts) rapidly (within 2h) incorporate matrix proteins and develop into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import rather than being involved in fusion of endoplasmic reticulum-derived peroxisomal vesicles. The revised manuscript describing these results has been submitted for publication in the Journal of Cell Biology. (Knoops et al., Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1. Submitted)
The initial phase of peroxisomal fission requires the peroxisomal membrane protein Peroxin 11 (Pex11p), which remodels the membrane, resulting in organelle elongation. We identified an additional function for Pex11p, demonstrating that Pex11p also plays a crucial role in the final step of peroxisomal fission: dynamin-like protein (DLP)-mediated membrane scission. First, we demonstrated that yeast Pex11p is necessary for the function of the GTPase Dynamin-related 1 (Dnm1p) in vivo. In addition, our data indicate that Pex11p physically interacts with Dnm1p and that inhibiting this interaction compromises peroxisomal fission. In particular using electron tomography, we were able to show that, although it seemed that more peroxisomes were present in a dnm1 deletion strain that overexpressed Pex11p, they are all connected via small neck-like membrane connections. Finally, we demonstrate that Pex11p functions as a GTPase activating protein (GAP) for Dnm1p in vitro. Our work identifies a previously unknown requirement for a GAP in DLP function. This results have been published in the Proceedings of the National Academy of Sciences. (Williams et al. (2015) The membrane remodeling protein Pex11p activates the GTPase Dnm1p during peroxisomal fission. Proc Natl Acad Sci U S A. 2015 May 19;112(20):6377-82.)
WP3. Analysis of in vitro peroxisome formation.
I have visited the laboratory of Prof. Randy Schekman during spring 2013. This visit has been very inspiring and I repeated the experiments that were published before. However, the setup of their experiment, i.e. microsomes isolated from pex19 cells which would generate vesicles upon in vitro addition of Pex19, raises questions about the true mechanism of vesicle formation. Since I recently demonstrated that vesicles are already present in pex19 cells, the main question arises whether the vesicles in the in vitro system are already present during microsome isolation (only associated to the ER e.g. via Inp1 interaction) and thus are not the result of an actual budding event. At this moment we are optimizing conditions for visualization of these vesicles as well as normal mature peroxisomes in a cryo-electron microscopy approach like stated in WP3.