PEX11
Promotes Peroxisome Division Independently of Peroxisome Metabolism
By Xiaoling Li and Stephen
J. Gould
“This web page was created as
an assignment for an undergraduate course at Davidson College.”
Peroxisomes
are membrane bound organelles, containing peroxidase and catalase, that import
all of their protein and most of their lipid content. Peroxisomes contribute to many lipid metabolic pathways including
B-oxidation of fatty acids. Based on
evidence that peroxisomes have a direct protein import pathway, it seems that
peroxisomes undergo growth and division.
It
had been previously established by Li and Gould that defects in
peroxisomal fatty acid B-oxidation reduce peroxisome abundance in mammalian
cells. Hence, there was the possibility
of metabolic control of peroxisome division.
Furthermore, PEX11 proteins are implicated in the regulation of
peroxisome abundance. Studies in yeast,
human, rodent, and protozoan forms of PEX11 have supported the notion that loss
of PEX11 causes a reduction in peroxisome abundance and PEX11 overexpression
causes an increase in peroxisome abundance.
These previous results would point to a direct role of PEX11 in
peroxisome division. It has recently
been proposed by Roermund et al. that the role of PEX11 in peroxisome division
is only a secondary function while its role in oxidation of medium chain fatty
acids (MCFAs) is its primary function.
In
this paper, Li and Gould test the response of PEX11 under various conditions in
mammalian, yeast, and mice cells. The
authors claim that their data suggest a revised role of PEX11—PEX11 plays a direct
role in peroxisome division and the loss of this protein inhibits peroxisome
metabolism indirectly.
Figure
1
illustrates that overexpression of human PEX11B induces peroxisome abundance in
a multistep process. PEX11 has two
forms, PEX11B and PEX11a, that are both integral peroxisomal membrane proteins
(PMPs). In this experiment wild type
human skin fibroblasts were injected with a PEX11Bmyc-containing plasmids. The cells were processed at varying time
points, 1.5, 4.5, and 48 hours, in order to determine the proliferating
activity over time. At each of the
three time points, the cells were visualized by immunofluorescence with
antibodies to the myc epitope or PEX14.
PEX14 is an endogenously expressed PMP.
From the results, it appears that after 1.5 hours PEX11B is
detected. After 4.5 hours, the
peroxisomes elongate and after 48 hours peroxisome number increases
noticeably. By using two different
antibodies, the myc which detects only those cells that take up the plasmid and
the PEX14 antibody which is endogenously expressed, this figure is well
controlled. This figure clearly shows
the presence and elongation of peroxisomes.
Also, cell formation is obvious after 48 hours.
The
authors next set out to determine the specificity and extent of PEX11B induced
peroxisome division. Normal human
fibroblasts were transfected with PMP34myc or PEX11Bmyc expression
vectors. After incubation, the cells
were processed for immunofluorescence using antibodies specific for the myc
epitope tag and PEX14. Figure 2
shows the results. According to the bar
graph in part A, peroxisome abundance is noticeably greater in the cells
overexpressing PEX11Bmyc compared to the untransfected cells and the cells with
the PMP34myc vector. Using both
untransfected cells and cells transfected with a general PMP provides two
controls. Representative cells are
shown in parts B-E after immunofluorescence with the two antibodies. Again, the cells expressing PEX11Bmyc (D and
E) had a great increase in the number of peroxisomes compared to cells
expressing PMP34myc. The authors
indicate the literature shows that when other cells expressing unrelated PMPs
(besides PMP34) were also myc tagged and monitored for peroxisome abundace,
this overexpression had no effect on peroxisome abundance. This figure shows that the increase in
peroxisome abundance induced by PEX11B expression reflects a specificity of
PEX11B and is not a consequence of PMP overexpression. The data is well controlled by using a
PMP34myc in addition to PEX11B and by marking with an antibody to the myc and
PEX14 (as in Figure 1). I do wonder why
the untransfected cells were not labeled with the two antibodies and the peroxisome
abundance shown as in parts B-E. I
suppose in theory this would not show anything drastically different compared
to B and C. While one could argue that
cells expressing other PMPs should be used in this experiment, the authors
refer to other papers reporting that this would not give contradicting
results. The data does support that
overexpression of specifically PEX11B increases peroxisome abundance in wild
type human skin fibroblasts.
Roermund
et al. hypothesized that the primary role of PEX11 is in MCFA oxidation and
peroxisome division is an indirect effect of this oxidation. Therefore, a functional peroxisomal
B-oxidation pathway is essential if PEX11 is to have a role in peroxisome
abundance. To test this hypothesis, Li
and Gould determined if PEX11 mediated peroxisome division could occur in cells
lacking a functional peroxisomal B oxidation pathway. The human cell line PBD005 was chosen as this lacks all
peroxisomal metabolic function but still contains peroxisomes. PDB005 cells were transfected with PMP34myc
and PEX11Bmyc. The results are shown in
figure 3. Again the bar graph
shows that cells overexpressing PEX11Bmyc have thirty times more peroxisomes as
compared to untransfected cells and cells transfected with PMP34myc. Representative cells (shown in B-E) labeled
again with antibodies to the myc tag and PEX14 tag, show more abundance of
peroxisomes in PEX11Bmyc cells than PMP34myc cells. The same controls were employed as in figure 2. From this data, it is concluded that the
peroxisome proliferating activity of human PEX11B is not dependent on
peroxisomal B-oxidation activities.
Since a cell line that is defective in all peroxisomal metabolic
activities was used, it is concluded by the authors that the peroxisomal
proliferating activity of human PEX11B is independent of all peroxisomal
metabolic activities. This refutes
Roermund et al.’s hypothesis. Since the
PDB005 cell line was so appropriately chosen and multiple controls were done,
it would appear that the data does support the author’s refute of Roermund’s claims.
Peroxisomes
are the only site of fatty acid B-oxidation in S. cerevisiae. Li and Gould mention that previous studies
have demonstrated peroxisome abundance increases when S. cerevisiae
cells are shifted from glucose dependent growth to growth on fatty acids. The abundance of peroxisomes was examined in
several S. cerevisiae cell lines with varying inserts and on varying
media. In figure 4, parts A and
B, the range of peroxisome abundance is established. The S. cerevisiae laboratory strain BY4733 was transformed
with a GFP containing plasmid and Gal promoter. When shifting the same cells from glucose media to the fatty acid
media oleic acid, there was a marked increase in peroxisome number (A and B). In parts C-G, the cell line XLY1 is used. This cell line contains the GFP plasmid with
Gal promoter. When shifting cells from
glucose to galactose, there is an immediate increase in peroxisome abundance (C
and D). The galactose promoter is no
longer repressed. Galactose induced
PEX13 (E) and Ypr128c (F) plasmids did not increase peroxisome abundace
compared to the empty vector (D).
However, galactose induced expression of PEX11 (G) did increase
peroxisome abundance up to the level observed on oleate media. I find it curious that the plasmid contained
PEX11 and not PEX11B. I am assuming
that since this has shifted from human cells to yeast, there are no alpha and
beta forms. But it would be nice if the
authors clarified this point. Finally,
in figure 4, a different strain of S. cerevisiae cells, XLY2, were
transfected with similar plasmids containing a gal promoter. The galactose induced expression of PEX13
had no effect on peroxisome abundance.
Again, the galactose induced expression of PEX11 increased the
peroxisome abundance to levels similar in oleate grown BY4733 cells and XLY1
cells with PEX11.
From these results, the authors make
the claim that in the absence of fatty acids from the growth media, it is
extremely unlikely that the peroxisomal fatty acid B-oxidation pathway was engaged. This then leads to the conclusion that
peroxisomal proliferation in S. cerevisiae is independent of the B-oxidation
pathway. From reviewing this data, I do
believe that the author’s have a justified reason to make these claims. There were multiple controls regarding media
(both with and without fatty acids), cell line, and plasmid insert. The results consistently showed that
peroxisome abundance increased with the PEX11 insert, regardless of cell line,
up to the extreme level established with growth on the oleate media. Consistent results and appropriate controls
lead me to believe the author’s conclusions.
Once
studying the role of PEX11 in human and yeast cells, the authors then focus on
mouse cells. Li and Gould test the
effect of the loss of PEX11B on peroxisome metabolism. Mouse embryonic fibroblasts from PEX11B+/+
and PEX11B-/- cell lines were cultured on serum free media and then processed
for immunofluorescence using antibodies for peroxisomal enzyme catalase (a
matrix marker) and PEX14. Figure 5, part A, shows that there is a
decrease in peroxisome abundance in PEX11B-/- mouse fibroblasts compared to
wild type cells. The results for
culturing the fibroblasts in normal conditions and in serum free media are
shown for both the wild type and PEX11B-/-.
This is well controlled, yet when taking into account the error bars, I
do wonder if there is a significant difference between the wild type and
PEX11B-/- cells. In the next part, wild
type and PEX11B-/- mouse fibroblasts were cultured in serum free media and then
visualized each with PEX14 antibody and matrix marker enzyme catalase. The results show that the abundance of
peroxisomes in wild type cells was twice that in PEX11B-/- grown under identical
conditions.
The authors reason that if PEX11B
functions primarily in fatty acid oxidation, then peroxisome abundance should
the same in wild type and PEX11B-/- cells when these cells are grown on serum
free media—devoid of lipids and substrates of fatty acid oxidation. On the other hand, if PEX11B’s primary
function is peroxisome division, peroxisome abundance should be reduced in
PEX11B-/- compared to wild type cells.
The data in figure 5 indicate just this. The loss of PEX11B appears to affect peroxisome abundance
independently of peroxisomal metabolism.
I find the data in this figure to, yet again, support the author’s
claims. My only question is with the
bar graph and the idea of statistical significance. But since Molecular Biologists often do not worry with this, the
immunofluorescence data that is well controlled seems sufficient to support the
author’s claims.
I
found this paper to be well written with sufficient data to support the
author’s claims. The data was presented
in a straightforward manner with multiple controls in each experiment. My only concerns are that, first, the
authors often refer to the role of PEX11 when drawing conclusions from a set of
data while the data only refers to the role of PEX11B. Second, the paper never addressed the role
of PEX11a. I wonder if this is unique
to human cells and if the role of PEX11a could be exclusive to peroxisomal
metabolism. The paper provides evidence
that
à overexpression of PEX11B
increases peroxisome abundance in wild type human skin fibroblasts
à overexpression of PEX11B
increases peroxisome abundance in a human cell line defective in all
peroxisomal metabolic functions
à overexpression of PEX11 in S.
cerevisiae increases peroxisome abundance in lipid free medium
à PEX11B-/- cells have only
half the peroxisome abundance as wild type cells when grown in serum free
medium
I
found the data adequate to support the claim that PEX11 (PEX11B in humans and
mice) proteins promote peroxisome division regardless of the metabolic pathway
in peroxisomes.
The
role of PEX11 needs to be characterized both in the metabolic pathway and in
peroxisome division.
The
authors mention that a possible way in which multiple peroxisomal metabolic
pathways could be damaged in PEX11 mutant cells is if the loss of PEX11
proteins alters the physical properties of the peroxisome membrane. Hence, I propose that an experiment be
performed to characterize the membrane of a wild type peroxisome, a PEX11
mutant, and another mutant such as PEX13.
This would have to be done in whichever organism the appropriate mutants
exist. FRAP (Fluorescence Recovery
After Photobleaching) is one such method that can be employed to determine if
these proteins are able to move within the peroxisome membrane.
It
is known that PEX11 is a membrane bound protein encoded by the PEX11 gene. The question is if the protein is an
integral membrane one and if so, which part of the protein interacts with the
molecules involved in the fatty acid metabolic pathway. First, after sequencing the gene by the
Sanger Method, the protein sequence can be deduced. Then a Kyte-Doolittle analysis can be performed to predict the
hydrophobic and hydrophilic regions of the protein. If it is an integral membrane protein, then the graph should span
the X-axis. To determine if the
cytoplasm or peroxisome portion of the PEX11 protein is where initiation of
metabolism occurs, the yeast two hybrid system could be employed. The initiator protein of the fatty acid
B-oxidation cycle should interact with either the part of the PEX11 that faces
the cytoplasm of the cell or the part of the PEX11 that faces the lumen of the
peroxisome. If Beta gal is used as a
reporter, then the cells that turn blue will contain the part of the PEX11 that
interacts with the metabolic pathway. I
suppose that ideally, it would be best to crystallize the protein interacting
with the molecule in the metabolic pathway.
But this is a bit more arduous.
The
authors also ponder the role of PEX11 proteins in peroxisome division. Specifically, Li and Gould mention that
peroxisome division may be sensitive to PEX11 concentrations in the peroxisome
membrane. An experiment could be performed
to take concentrations of the PEX11 membrane protein at different points in the
peroxisome division.
Perhaps
during peroxisome division, a part of the peroxisome membrane dissolves such as
when a bud pinches off. To investigate
membrane continuity during division, FLIP (Fluorescence Loss in Photobleaching)
can be employed. The investigator would
have to isolate cells at varying stages of peroxisome division in order to
understand the changes in the membrane and the proteins within the
membrane.
For questions or comments contact elsellars@davidson.edu