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PEX11 promotes peroxisome divison independently of peroxisome metabolism
Xiaoling Li and Stephen J. Gould
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
Journal of Cell Biology (2002) 156, 243-651.
Summarize and Critique:
Peroxisomes are membrane bound organelles in eukaryotic cells that serve as sites for some metabolic reactions. The specific pathway addressed in this paper refers to the b-oxidation of fatty acids, especially medium chain fatty acids (MCFAs) and very long chain fatty acids (VLCFAs). Peroxisomes contain several oxidases for the oxidation of organic fatty acids and catalase for the degradation of hydrogen peroxide (Lodish et al., 2000).
Since peroxisome division is not well understood, researchers have been eager to link peroxisome formation to other cellular processes (Chang et al. 1999). One protein, PEX11 (a and b human forms), has received attention recently after several groups (Abe and Fujiki, 1998; Abe et al., 1998; Schrader et al., 1998) proved that PEX11 overexpression in human fibroblasts results in abnormally high cellular peroxisome content. Striving to link this protein to other metabolic processes, van Roermund et al. (2000) asserted that peroxisome division is only a secondary implication of the function of PEX11 in MCFA oxidation - a hypothesis that implicates the existence of an unidentified signaling molecule responsible for peroxisome division. Considering this history, Li and Gould sought to prove that peroxisome division and peroxisome metabolism are not necessarily interconnected functions. This revised model implies that a PEX11 deficiency stalls peroxisome metabolism.
Using a line of wild-type human skin cell fibroblasts (line GM5756), the researchers microinjected a plasmid (pcDNA3-PEX11bmyc) that prompts the overexpression of PEX11b protein. In figure 1, the transfected fibroblasts are processed for varying time lengths and injected for double indirect immunofluorescence with two different types of antibodies: anti-myc and anti-PEX14. PEX14 is an endogenously expressed peroxisomal membrane protein (PMP) believed to be a peroxisome membrane receptor docking site (Urquhart et al., 2000). Within 24-48 hours after plasmid injection, the cellular peroxisome content was noticeably increased.
To quantify the increase in cellular peroxisomes, the researchers transfected GM5756 cells with either pcDNA3-PEX11bmyc or pcDNA3-PMP34bmyc. PMP34 is a human peroxide membrane protein homologous to a yeast protein essential for MCFA b-oxidation (Wylin et al., 1999), unrelated to PEX11. Again, cells were processed for indirect immunofluorescence with anti-myc and anti-PEX14 antibodies. The samples were resolved by confocal fluorescence microscopy and the number of discreet peroxisomes per section (pps) was quantified and reported in figure 2. Where untransfected and PMP34myc transfected cells had similar pps values, cells overexpressing PEX11bmyc exhibited a 1,000% percent increase in peroxisome content. 10 other forms of human myc-tagged peroxisomal proteins were also overexpressed and investigated for peroxisome abundance, but only PEX11b expression induced high peroxisome content. The researchers deduce that not any PMP, but specifically PEX11b is responsible for peroxisome abundance.
To discount the hypothesis of van Roermund et al. (2000), the researchers recruited the use of a new fibroblast line, PBD005. These Zellweger syndrome cells have peroxisomes but are missing all peroxisomal metabolic functions. Performing the same experiment on these cells as the GM5756 cells in figure 2, there was no significant difference in peroxisomal abundance. Figure 3 reports low levels of peroxisomes in untransfected and PMP34myc transfected cells compared to the drastically increased number of peroxisomes in cells overexpressing PEX11bmyc. When compared to figure 2, these data denote independent mechanisms for peroxisome division and peroxisome metabolism.
Having demonstrated the how PEX11 affects peroxisome divisions regardless of peroxisome metabolism in human fibroblasts, the researchers move to prove the same contention in yeast cells. A strain of S. cerevisiae (BY4733) were transformed with a plasmid that consitutively expresses green fluorescent protein designed to be incorporated into peroxisomes (GFP/PTS1). Panels A and B in figure 4 are controls for their yeast strain (BY4733) which demonstrates the average numbers of peroxisomes per cell of yeast grown in glucose (A) and oleic acid (B). The shift in peroxisome number in panel B is expected as a consequence of metabolism shift favoring growth on fatty acids. To create a new test strain, the researchers deleted the normal PEX11 gene from BY4733 and inserted the plasmids pPGK1-GFP/PTS1 and pRS425/GAL1. This new strain, XLY1, was galactose induced and transformed with many different PMPs including PEX13p, Ypr128Cp, and PEX11p. Of these PMPs, only PEX11 indicated a shift in the number of peroxisomes per cell, similar to ths shift seen in oleate-grown cells. The researchers also tested PEX13p and PEX11p XLY2 cells, a POX2 derivative encoding peroxisomal acyl-CoA and again, only the PEX11 expressing cells exhibited a shift in peroxisome number.
To prove specifically that PEX11 involves peroxisome division independently of MCFA oxidation, the researchers used mouse fibroblasts to dispel doubts that PEX11 proteins have multiple and unrelated functions in both division and metabolism. Figure 5 compares the relative peroxisome density in PEX11b-/- and PEX11b+/+ cells cultured in serum-free medium (without substrates necessary for peroxisomal fatty acid oxidation) and indirectly immunofluoresced with anti-PEX14 antibodies and matrix marker enzyme catalase. In both cases, the cells expressing PEX11b had a greater peroxisome abundance than the cells with deleted PEX11b. Since neither cell type was undergoing any sort of peroxisomal fatty acid oxidation in the serum-free medium, the imbalance in peroxisome abundance between PEX11b-/- and PEX11b+/+ cells implicates independent mechanisms of metabolism and division.
In critical evaluation, the paper is well written, logical, and reasonably supprted. The most impressive aspect of the paper was the way each experiment was repeated with many different approaches. Every immunoflorescence was tested by at least two differnt markers (anti-myc, anti-PEX14, matrix marker enzyme catalyase) and three different yeast strains were tested in a total of three different environments with 4 different types of transfections. Whereas the data from human fibroblasts may have been enough to prove the hypothesis, the researchers graciously conducted slightly different variations on the same experiment with two other organisms, yeast and mice.
There are a few loose ends that detract from the paper, but do not ultimately discount the hypothesis. For example the researchers claim PEX11b and PMP34 are unrelated PMPs, but do not offer a citation or data. Also, the paper does not consider the possible functional changes caused by the addition of the myc epitope, especially since PEX11 has been shown to form homodimers (Marshall et al., 1995). With both PEX11 termini facing out into the cytoplasm (Passreiter et al., 1998), the addition of epitopes could alter shape and thus function, especially since the paper proposes precisely that PEX11 alters membrane structure and dynamics to affect peroxisome division.
Future experiments:
In this paper, the researchers very directly contradicted the hypothesis of van Roermund et al. (2000) and propose a new model. Both versions implicate a different factor which may be the determining factor in testing the validity of these theroies. For example, van Roermund et al. (2000) proposes the existence of a signaling molecule while Li and Gould (2002) propose an alteration in membrane structure or dynamics. Van Roermund's group is then challenged to find their receptor while Gould's group has the difficult task of proving membrane altercations.
Since Li and Gould (2002) have done the best job of functionally promoting their hypothesis thus far, I would design an experiment to find their membrane alterations. One approach includes FRAP to trace the movement of proteins within the peroxisome membrane. Since FRAP can follow the movements of a molecule in time, the next experiment is to systematically tag each PMP with a fluorophore and compare movements of the protein in the presence and absence of PEX11. Those proteins with altered locations or mobilities in the absence of PEX11 can also be tested (as in this paper) for implications in division or metabolism. With enough work (by a very unfortunate graduate student) a PMP network can be devised to explain the specific function - metabolism or division.
It seems as though the researchers are already preparing to investigate these proteins, initially with VPS1, a dynamin-related protein recently proven necessary for peroxisome division (Hoepfner et al., 2001). Van Roermund's group should work quickly in finding their signaling molecule generated by metabolite flux - if it exists at all.
Lodish, Berk, Zipursky, Matsudaira, Baltimore, and Darnell. Molecular Cell Biology. 4th ed. New York: Freeman, 2000.
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