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PEX11 promotes peroxisome division independently of peroxisome metabolism
Xiaoling Li and Stephen J. Gould, 2002. J. of Cell Biol., 156 (4): 643 - 641
Peroxisomes are membrane-bound organelles that aid in several pathways in lipid metabolism, including the b-oxidation of fatty acids. PEX11 proteins are involved in the regulation of peroxisome abundance. Previous studies have proposed two separate models for the role of PEX11 proteins in peroxisome division, a poorly understood phenomenon. The first model suggests that peroxisomes play a direct role in division, while the second model indicates that the role of PEX11 in peroxisome division is a secondary effect of its primary role in the oxidation of medium chain fatty acids (MCFAs). This project sought to determine which of these models provides the most accurate representation of the role of PEX11 in peroxisome division.
PEX11 is expressed in two forms in humans, PEX11a
and each serves as an integral peroxisomal membrane protein (PMP).
Figure 1 is a set of immunofluorescence microscopy slides indicating the
effect of the overexpression of PEX11b
in normal human fibroblasts. The
technique and results had been previously published.
Plasmids containing the PEX11bmyc
construct were microinjected into the cells, and then the cells were probed with
the anti-myc antibody and viewed at specific time intervals to show the increase
in abundance of peroxisomes. These
slides (A, C, and E) were compared with slides of the same cells probed with
anti-PEX14 antibody (B, D, and E, offered as a control).
PEX14 is an endogenously expressed PMP, and these slides show the
location of the undivided peroxisomes within these cells.
After 48 hours, abundance of peroxisomes had increased as compared to the
control. Figure 1 then
establishes two points: PEX11bmyc
is indeed detected in peroxisomes of normal morphology and abundance (see A and
B) and PEX11b
does induce an increase in peroxisome abundance.
Figure 2 quantifies the peroxisome abundance in normal human fibroblast cells transfected with the PEX11bmyc construct in expression vectors (leading to its overexpression) and compares this to the number of peroxisomes in untransfected cells probed by a-PEX14 and cells transfected with a plasmid containing PMP34, a different PMP, also labeled with myc and probed by a-myc. Indeed, overexpression of PEX11b does lead to an increased abundance of peroxisomes—an actual increase of 1000%. Photos of representative slides easily show examples of this abundance. Peroxisome abundance was also examined in cells overexpressing ten other myc-tagged forms of PMPs, and the overexpression had no effect on peroxisome abundance (data unpublished).
The authors next sought to determine whether PEX11-mediated peroxisome division occurs in cells that lack a functional peroxisome b-oxidation pathway, testing a previously proposed hypothesis that this pathway is needed for PEX11 proteins to have any effect on peroxisome division. A cell line defective in numerous peroxisomal metabolism activities, including the peroxisomal fatty acid b-oxidation pathway, was transfected with either a plasmid containing PEX11b-myc construct or a plasmid containing the PEX34-myc construct. The graph in Figure 3 shows that overexpression of PEX11b does increase peroxisome abundance, as compared to controls, by a factor of thirty. The cell line used is also defective in importing any peroxisomal matrix enzymes; thus the mechanism of peroxisome proliferation by human PEX11b is independent of peroxisomal metabolic pathways. Again, photos of slides of the transfected cells are shown to further illustrate that overexpression does lead to an increase in peroxisome abundance.
To illustrate that the PEX11 mechanism of peroxisome proliferation in yeast did not require fatty acid b-oxidation, the authors overexpressed PEX11 in S. cerevisiae cells grown on lipid-free medium. They first illustrated in Figure 4A and 4B that normal cells have more peroxisomes when grown in the presence of fatty acids. They detected these numbers by transfecting cells with a plasmid containing GFP that could be imported into the peroxisome lumen and counting the number of peroxisomes. In Figure 4C and 4D, the chromosomal copy of PEX11 was deleted before transfection of the GFP plasmid. A plasmid containing the GAL1 promoter was also transfected into these cells. Figure 4D becomes the control for the next few parts of the experiment, as it shows the number of peroxisomes per cell when PEX11 is deleted and the cells are grown on galactose. It is important to show what occurs in the cells with the “empty promoter.”
When two other forms of PMPs were overexpressed using
the GAL1 promoter, no change in peroxisome abundance was noticed (Figure 4E and
F). However, an increase in
peroxisome abundance was seen when PEX11 was overexpressed using the GAL1
promoter (Figure 4G). These
levels of peroxisome abundance were very similar to the levels observed in the
cells grown on the fatty acid medium. They
also repeated these same experiments using cells that cannot do fatty acid b-oxidation.
In these cells expressing the GAL1-induced PEX13, peroxisome abundance
did not increase, whereas the number of peroxisomes in cells expressing the
GAL1-induced PEX11 did increase with numbers similar to the increase shown in
Figures 4B and 4G. Thus, PEX11
overexpression does lead to an increase in peroxisome abundance.
PEX11 in both humans and yeast promote
peroxisome division independently of fatty acid b-oxidation.
However, when yeast PEX11 is not present, the oxidation of MCFA is
reduced. Thus, PEX11 must have two
independent functions, or its ability to reduce MCFA oxidation is an indirect
effect of its ability to promote peroxisome proliferation.
The authors chose another model, the mouse, to test these hypotheses.
The graph in Figure 5 shows peroxisome abundance in mice homozygous for
gene and in mice homozygous for the PEX11b
deletion. The cells were grown in
medium devoid of lipids and the substrates needed for the peroxisomal fatty acid
pathway or in medium in normal conditions.
The number of peroxisomes in cells homozygous for PEX11b
was the same in both normal conditions and conditions lacking lipids and fats.
These numbers were higher than those obtained from cells lacking PEX11b.
affects peroxisome proliferation independently of fatty acid peroxisomal
metabolism in mice.
This paper first demonstrates that human PEX11b overexpression is sufficient to promote peroxisome division, although the authors state that PEX11 is sufficient. I do not agree that PEX11 is sufficient. Only one form of it is sufficient and that is the b form. To accurately claim that PEX11 is sufficient, both forms should have been tested. What does PEX11a do? Does it play a role similar to that of PEX11b? Does a combination of the two forms contribute to PEX11’s role as a PMP? Furthermore, while the authors show one representative example of PMPs in Figure 2, I think that their case would have been more conclusive if they had published the results surrounding at least one or two other PMPs.
A second point is that for Figure 1, there were no slides containing cells without plasmids. Would this not be an ideal control? They could have probed this slide with an antibody against another PMP. Another slide would be beneficial to show the relative locations and abundance of the myc protein prior to transfection. Myc is a transription factor endogenous to human cells and should be located in the nucleus when probed with a-myc. Establishing its location prior to transfection could help distinguish where this endogenous protein is located as opposed to the location of the myc-tagged protein. This control should have ideally been done in all experiments.
Finally, the authors demonstrate that peroxisome abundance increases even when the peroxisome itself cannot perform any metabolic functions. This is the extreme, as the cells used could perform no functions. However, would the results still be the same if they could perform a few metabolic functions? Maybe a range of tests from cells that could perform all metabolic functions to cells that performed no metabolic functions would have been beneficial.
Overall, the paper presents good controls and adequately explains its reasoning. As far as the data are concerned, I am convinced that human PEX11 does play a direct role in peroxisome proliferation and that its role in MCFA metabolism is secondary to its proliferative role.
There are a few other things to explore however.
What happens when PEX11b is not expressed in human cells? The authors illustrate overexpression, but there is no comparison to no expression or even underexpression in terms of peroxisome proliferation. Deleting the chromosomal copy and looking at peroxisomal abundance in these cells compared to wildtype expression and overexpression may answer the question of necessity. Is PEX11b necessary for peroxisome division?
If peroxisomes proliferate regardless of the presence of PEX11b, then what are the other factors involved in peroxisome division? Testing the presence and absence of other enzymes or proteins and the effects on peroxisome proliferation would help answer questions in this discussion.
The next question to address concerns the role of PEX11 in yeast. PEX11 appears to be a requirement for MCFA oxidation in yeast. Once way to test this is to determine PEX11’s placement in the MCFA oxidation pathway. If some information of this pathway is known, then one could see where the pathway stops when PEX11 is absent. What molecules does it interact with? Researchers could do assays where peroxisome membranes are mixed with various enzymes and other proteins known to be part of the pathway and then total protein could be run on a gel and probed with antibodies against PEX11. If molecular weights are altered in specific lanes, then the extra protein added to the assay in that lane may be binding to the PEX11 protein. Different assays such as these could provide clues to PEX11’s role in MCFA oxidation.
Finally, what exactly is the role of PEX11 in peroxisome division? A few thoughts arise. As an integral PMP, it must receive some sort of signal from either outside or inside the peroxisome. When there are more of these proteins are present, more of the signal can bind, signaling more peroxisome division and proliferation. By studying the structure of PEX11 in the peroxisome membrane (via hydropathy plots) one could distinguish whether a signal is received from inside or outside of the peroxisome. The extra-peroxisomal part of the protein could be removed and then functional assays could be performed to see if the peroxisomes are still able to divide. The opposite could be done with the intra-peroxisomal part of the protein.
© Copyright 2002 Department of Biology, Davidson College, Davidson NC 28035
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