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review
paper |
PEX11 promotes peroxisome
division independently of peroxisome metabolism
Xiaoling Li and Stephen J.
Gould
The Journal of Cell Biology,
Volume 156, Number 4, February 18, 2002 643-651
Peroxisomes are involved in lipid metabolism, including the pathway for the b-oxidation of fatty acids. Peroxisome growth through the uptake of various proteins and lipids results in the division and thus proliferation of new peroxisomes. PEX11 is a peroxisomal membrane protein that has been implicated in peroxisome division and may also be involved in peroxisomal b-oxidation of fatty acids. The actual mechanisms behind peroxisome division are poorly understood; however, it has been shown in yeast that an increase in pex11 expression correlates with an increase in peroxisome abundance. Likewise, repression of pex11 was associated with a reduction in peroxisome abundance. Human, rodent, and protozoan forms of this gene act in a similar fashion. PEX11-a and PEX11-b are two forms of PEX11 expressed in humans and act as integral peroxisomal membrane proteins (PMPs).
van
Roermund et al. have proposed that
pex11 is indirectly involved in the division of peroxisomes through its role in
fatty acid oxidation. They suggest that the oxidation of medium chain fatty
acids (MCFAs) may stimulate a molecule to signal peroxisome division. Li and Gould explore the hypothesis
formulated by van Roermund et al.
regarding the function of pex11 in both yeast and mammalian cells. Contrary to the hypothesis, their findings
suggest that PEX11 proteins are directly involved in the division of
peroxisomes since peroxisome division was carried out by PEX11 sans peroxisome metabolism. This paper also demonstrates the indirect
relationship between a loss of pex11 and inhibition of peroxisome
metabolism. This connection was drawn
by examining the loss of pex11-b in mice and the ensuing
decrease in abundance of
peroxisomes.
Temporal
expression of PEX11-b was analyzed in human skin
fibroblasts. Plasmids containing both
pex11-b and the c-myc tag were
injected into these wild-types cells.
Fluorescently-labeled 9E10 antibodies detected PEX11-b expression as it progressed through three
kinetically-distinct steps between 1.5 hours and 48 hours. These figures illustrate the presence of
PEX11 within the peroxisomes. The boxes
within Figures
1a, 1c, and 1e are simply magnifications of that figure. Figures 1a and 1b reveal normal peroxisome abundance 1.5 hours after transfection. However 4.5 hours after introduction of this plasmid into the fibroblast, the peroxisomes lengthen (Figures 1c and 1d). This elongation is particularly evident in the magnified portion of Figure 1c. Perhaps this elongation precedes peroxisome division, because Figures 1e and 1f exhibit increased peroxisome abundance 48 hours after microinjection of pex11-b. This figure suggests that the introduction of PEX11-b initiates peroxisome division through three stages. It would have been a nice control if the authors had proven that the PEX11 protein functioned normally with the c-myc tag attached. The protein may have been bulky or its confirmation may have been altered by the presence of c-myc. To ensure that the c-myc tag was expressed properly, a Western Blot of PEX11-b and PEX11-b/c-myc probed with an antibody to PEX11-b would have been beneficial. Both proteins should be detected at roughly the same molecular weight on this immunoblot.
A
second experiment involved ascertaining the specificity of peroxisome division
induction. Human fibroblast cells were
transfected with pex11-b/c-myc, pmp34/c-myc, or
nothing. (pmp34 is the human homologue
to a peroxisomal yeast gene.) These
cells were allowed to grow for 2 days and then were probed with either a
fluorescently-labeled a-myc antibody or a-PEX14 antibody. a-myc antibody labels the a-myc tag; however PEX14 is an unrelated, yet
endogenous PMP and would label the PEX14 constitutively expressed in
peroxisomes. Confocal fluorescent
microscopy was used to count the number of peroxisomes. Figure 2a displays the number of peroxisomes
found in three different cell types.
The untransfected cell serves as the control and reveals the normal
peroxisome abundance. The cell
transfected with the pmp34/c-myc construct has a similar abundance. However, the pex11-b/c-myc transfection seems to have significantly
increased the peroxisome's presence in these fibroblasts. Figure 2b, c, d, and e show some of the
actual fluorescently-labeled fibroblasts.
Figure 2b confirms the presence of the pmp34/c-myc construct in the
cell. The a-myc antibody was used here to label
peroxisomes that contained the construct.
Figure 2c contains the same construct, but was labeled with a-PEX14.
Figure 2d was injected with pex11-b/c-myc and was probed with
a-myc. This figure has more peroxisomes
present than Figure 2b. Figure 2e has
the same pex11-b/c-myc construct, but it is
labeled with the antibody to PEX14.
Figures 2d and 2e exhibit a higher abundance of peroxisomes than Figures
2b and 2c. In fact, Figures 2b and 2c
display the baseline amount of peroxisomes as demonstrated by Figure 2a. The authors conclude that the increased
abundance of peroxisomes coincides with the expression of PEX11-band is not induced by PMP
overexpression. However, this
experiment relies on the assumption that the constructs are fully functional
and that the antibodies are indeed specific.
Both constructs could be proven functional through a Western Blot
similar to the one described above. The
9E10 antibody has been proven in previous experiments to be specific; however
the PEX14 antibody may not have been. A
Western Blot in which all human fibroblast proteins were present would be nice
to ensure the specificity of a-PEX14 antibody.
van
Roermund et al. also hypothesized
that the promotion of peroxisome division by PEX11 results from the role of
these proteins in peroxisome metabolism, specifically MFCA oxidation. Cell lines deficient in peroxisomal
metabolic functions were used to determine if a dysfunctional peroxisomal b-oxidation pathway would have any bearing on
the PEX11-mediated proliferation of peroxisomes. The results in Figure 3 are similar to those in Figure 2. Figure 3a shows that the pex11-b/c-myc construct led to more peroxisomes than
the untransfected cells and those that were transfected with pmp34/c-myc. This figure is also important because it
demonstrates the effects of s lsck of all peroxisomal metabolic functions on a
cell. These untransfected cells have a
reduced abundance of peroxisomes compared to the untransfected fibroblasts in
Figure 2 with wild-type metabolic function.
Figure 3b serves as a control for Figure 3d since it depicts PMP34 and
PEX11-b respectively. These two figures were probed with
fluorescently-labeled a-myc. An antibody for PEX14 probed Figures 2c
(pmp34/c-myc construct) and 2e (pex11-b/c-myc construct). Figure 3 illustrates the abundance of
peroxisomes in the fibroblasts that overexpress PEX11-b, even in the absence of peroxisomal
metabolic activities.
A
fourth experiment involved switching from humans to yeast in order to determine
that PEX11-mediated division of peroxisomes occurs without the b-oxidation of fatty acids and its concomitant
metabolites. In yeast, peroxisomes host
the site for fatty acid b-oxidation and are known to
proliferate when switched from media containing glucose to a fatty acid
medium. A yeast strain was created to
express GFP/PTS1 constitutively, where GFP would be transported into the lumen
of the peroxisome. The purpose of GFP
is the visualize peroxisomes within the yeast.
Figure 4a depicts yeast grown in glucose, whereas the yeast in Figure 4b
was grown in glucose and then shifted at midlog phase to minimal medium with
oleic acid. The yeast grown in the
fatty acid has more peroxisomes than the yeast cultured in glucose-dependent
medium. However, it was not the
addition of a fatty acid to the yeast that increased peroxisome number, rather
the change in medium. When yeast was
shifted from glucose- to galactose-containing medium, peroxisomes were more
abundant. For instance in Figure 4d,
the yeast are released from repression of adr1 (a peroxisomal protein
regulator) by glucose. Even though the
strain of yeast is different (Dpex11), the amount of
peroxisomes present in Figure 4c is comparable to Figure 4a. Figures 4e and 4f depict yeast grown in
galactose with a pex13 and Ypr128c transformation, respectively. These two genes code for PMPs and their
overexpression does not affect the peroxisome number more than just growing the
yeast in galactose medium. (The
peroxisome abundance is approximately the same in Figures 4e and 4f as it is in
4d.) When pex11 is transformed into the
same yeast strain and grown in the same medium as Figures 4c-4f, then more
peroxisomes are evident (approximately the same number as in Figure 4b.) The yeast in Figure 4b were grown in oleic
acid, but the quantitatively similar peroxisome division in Figure 4g were not
likely affected by any fatty acid b-oxidation substrates since
this yeast grew in minimal medium plus galactose. In a different approach, a yeast strain was used that encodes the
first committed step for the b-oxidation of fatty acids
(delete pex11, add pox1.) PEX13 did not
affect the abundance of peroxisomes (Figure 4h), whereas PEX11 was shown to
increase proliferation (Figure 4i) to the same degree as Figures 4b and 4g. A lot of variations and controls were
performed in this particular experiment.
The
conclusions that human and yeast PEX11 induced proliferation of peroxisomes
independent of peroxisome metabolism and that peroxisomal oxidation of MCFA is
decreased by the deletion of yeast PEX11 led to the question of whether PEX11
had multiple direct functions or acted indirectly on peroxisome
metabolism. A murine system with a
pex11-b homozygous deletion was
developed and studied to make this determination. Mouse embryonic fibroblasts from pex11-b+/+ and pex11-b-/- mice were compared and the
mice with pex11-b had more peroxisomes under
both normal and serum-free conditions than mice homozygous for the pex11-b deletion (Figure 5a). Figures 5b-5e depict mouse fibroblasts grown
in serum-free medium. Figures 5b and 5c
have different antibodies (a-PEX14 and a-catalase, respectively) but both depict
fibroblasts expressing PEX11-b. Figures 5d and 5e do not have as many peroxisomes because these
embryonic fibroblasts are pex11-b-/-. This figure shows that in mice, the effects of PEX11-b loss on peroxisome abundance are not
dependent upon peroxisomal metabolism.
How
PEX11 could alter membrane structure or dynamics? It is possible that PEX11 may be indirectly affecting peroxisomal
metabolism by altering the peroxisome structure or dynamics. If proteins in the membrane structure were
being altered, I would run a protein chip and see what was being induced and
repressed when PEX11 was induced on this macroarray. FRAP could be used to understand the movement of carbohydrates or
lipids in the peroxisomal membrane.
Known carbohydrates and lipids in the membrane would be tagged one at a
time with a fluorophore and then FRAP could be done before PEX11 is injected
into a cell and then after. If
differences were distinguished, then structural alteration would be a
possibility.
What
is the role of PEX11-interacting proteins in the division process? Researchers think that PEX11 proteins may
participate in VPS1-mediated peroxisome division or that they may act in some
other distinct process like coat-mediated peroxisome budding. First, I would use an mRNA microarray to
determine what genes were activated at the same time as pex11 and at different
time periods after pex11 induction since there are three kinetically-distinct
steps to peroxisome division. I may
also try an immunoprecipitation with a probe for PEX11 proteins. I would use a gentle detergent in order to
find coprecipitating proteins.
Coprecipitates could be labeled with fluorophores and then FRAP could be
used to better understand their role in peroxisome division. The yeast two-hybrid method could also be
used to find proteins that interact with PEX11.
Is
PEX11 activity affected by post-translational modifications? Post-translational modifications can be
determined by running a band shift assay, since SDS or other denaturing agents
are not required in this molecular weight resolver.
Is
peroxisome division affected by the concentration of PEX11? FISH could be used to determine at what mRNA
concentration the division of peroxisomes occurs.