To determine the overall function of Pex11 proteins, we explored the potential of heterologous Pex11 proteins to complement the pex11
Δ phenotype of S. cerevisiae
cells (). The Sc
Pex11 protein is more closely related to the heterologous Pex11 proteins than to the two other family members present in S. cerevisiae
, Pex25p and Pex27p (30
). Expression of the heterologous Pex11 proteins did not significantly change the number of peroxisomes in pex11
Δ cells. While they all localized to peroxisomes, only five of the eight proteins tested allowed the cells to consume oleic acid (). Noteworthy, among the five proteins complementing the oleate utilization phenotype, all but Hs
PEX11β contain at their C-terminus a −KXKXX motif known as ER-retrieval signal (31
). This motif may already indicate a connection of these peroxisomal proteins with the ER.
The three members of the Pex11 family in S. cerevisiae
were originally identified as factors controlling peroxisome number and function (16
). However, their individual contribution remained unknown. To discern their particular roles we expressed these proteins in single, double and triple mutants alone or in combinations, analyzed the ability of transformed cells to utilize oleate and evaluated the number of peroxisomes per cell ( and ). Our results provide evidence that each member of the Pex11 family holds a different function in the control of peroxisome number and metabolic activity. This suggests that in S. cerevisiae
, the delicate balance between Pex11p, Pex25p and Pex27p ensures a variable number of peroxisomes and guarantees that each cell is furnished with adequate peroxisomal metabolism.
Several characteristics suggest a role for Sc
Pex11p and close heterologous relatives in membrane remodeling. Its conserved localization () (3
), its abundance (16
) and the lack of a transmembrane domain but presence of an amphipathic helix (15
) might allow for exclusion or specific association of proteins or metabolites at the peroxisomal membrane. Amphipathic helices are thought to sense membrane curvature or to participate in membrane remodeling (33
). Thus, Pex11p might act as a sensor to determine the ability of the membrane to proliferate. Alternatively, a continuous gradient of Pex11p might allow for membrane protrusion. In both cases, accumulation of Pex11p at specific membrane sites with a precise form or lipid composition might influence the peroxisomal metabolism, a behavior that seems to have been conserved throughout evolution.
In the mere absence of Pex11p, cells are unable to utilize fatty acids. Interestingly, the additional lack of Pex27p allowed the cells to regain peroxisomal function (), suggesting a negative or competitive role for Pex27p. Consistent with this notion, the reintroduction of Pex27p into pex11
Δ cells and pex25
Δ cells reduced their ability to utilize oleate (). While overexpression of Pex25p in pex11
Δ cells led to the occurrence of elongated peroxisomal structures, the concomitant expression of Pex25p and Pex27p in these cells reduced the frequency of elongated peroxisomes (). The presence of functional peroxisomes in pex11
Δ cells and the occurrence of fewer cells with elongated peroxisomes in pex11
Δ mutants expressing Pex25p and Pex27p from plasmids are observations in agreement with a model in which Pex27p competes with Pex25p during the process of proliferation. The finding that Pex27p can partially substitute for Pex25p in de novo
formation of peroxisomes strongly supports a competition between these two proteins (), suggesting a similar role and a similar localization for both (18
). That this process is slow in the absence of Pex25p could be because of the fact that endogenous Pex27p is only present in small amount in wild-type yeast cells (18
). However, in contrast to Pex11p and Pex25p, overexpression of Pex27p does not lead to functional peroxisomes in cells lacking all three proteins (). In wild-type cells, overexpression of Pex27p showed only moderate influence on peroxisomal number. An explanation could be that Pex27p is only active when the balance between Pex11p and Pex25p is perturbed which could endanger the propagation of peroxisomes.
The negative effect exerted by Pex27p on peroxisomal function most likely takes place at the peroxisomal membrane. In wild-type cells, the presence of Pex25p or Pex27p at the peroxisomal membrane could locally alter the lipid-to-protein ratio, thereby enhancing the association of Pex11p with the membrane at this site. This in turn would result in Pex11p accumulation, membrane remodeling and proliferation at this exact site. The property of Pex11p to oligomerize (20
) might support a co-operative association with the peroxisomal membrane, which, in turn, could explain its function in proliferating peroxisomes already present in the cell.
In the absence of Pex11p and Pex27p, Pex25p is sufficient to provide the cells with functional peroxisomes. The occurrence of elongated peroxisomes (), strongly increased upon ectopic expression of Pex25p (), suggests that this protein triggers membrane elongation, a step essential to prime peroxisome proliferation. Pex27p might compete with Pex25p in the process of membrane association or at the level of protein interaction, e.g. with Pex11p. However, as there is no evidence for heteromeric interactions between Pex11 family members, the interplay between these proteins might rather rely on the interaction of each individual protein with lipids of the same (peroxisomal) membrane. We propose that the interaction between each member of the Pex11 protein family and the peroxisomal membrane has been conserved throughout evolution. This hypothesis fits the observation that heterologous Pex11 proteins localize to peroxisomes and compensate for the loss of peroxisomal function to various degrees () (3
Growth and division of existing peroxisomes and de novo
formation from the ER constitute the peroxisome biogenesis (10
). The protein Pex3p was previously described as an early peroxisome biogenesis factor and it was shown to be the initiating factor for peroxisome biogenesis from the ER (9
). While proliferation from existing peroxisomes could take place in the absence of the Pex11-family members, after loss of peroxisomes, Pex25p was required to generate wild-type levels of peroxisomes. Hence, we demonstrate that Pex25p acts in intimate co-operation with Pex3p and that both are equally required for de novo
formation. Similar results were obtained in the yeast Hansenula polymorpha
We present a model (), in which each one of the yeast Pex11 proteins holds an individual function in the formation of peroxisomes. In conclusion, (i) we demonstrate that Pex25p participates in membrane elongation of existing peroxisomes and in the initiation of de novo biogenesis from the ER, (ii) we provide evidence that Pex27p exerts an inhibitory or competitive function and (iii) we show that Pex11p only promotes the proliferation of peroxisomes already present in the cell.
A model for the function of Pex11p, Pex25p and Pex27p in peroxisome biogenesis