New insights in Pex11pβ-mediated growth and division

An important finding of this work exploiting the Pex11pβ-YFP fusion protein is the ob-servation that Pex11pβ-mediated growth (elongation) and division of peroxisomes fol-lows a multistep maturation pathway. Starting at pre-existing peroxisomes membrane extensions enriched in Pex11pβ are formed. These membrane tubules become positive for the so-called “early peroxins” Pex3p, Pex16p, and Pex19p required for peroxisomal membrane protein import (Figure 3.18 and Figure 3.19). Other PMPs, such as PMP70 or PMP22, and matrix proteins are absent from the tubules and only found in the globular (mature) peroxisomes giving rise to the membrane extensions (Figure 3.17, Figure 3.18 and Figure 3.22). As Pex11pβ appears to act as a dominant-negative mutant, it blocks the subsequent constriction/segmentation and division of the tubular membrane com-partment, which is e.g. observed after expression of Pex11pβ-Myc. The Pex11pβ-YFP mediated block or delay in the correct assembly of the constriction/division machinery is likely to inhibit the import of newly synthesized matrix proteins and PMPs into the membrane extensions as well. In contrast, proper import of matrix proteins and PMPs in the segmented, “beads on a string”-like peroxisomes is observed in controls expressing Pex11pβ-Myc. The import of newly synthesized matrix proteins into the “beads” was nicely shown by application of the HaloTag technology (Figure 3.22). Thus, it is pro-posed that the assembly of the constriction/division complex is a prerequisite for the proper assembly (or activation) of the protein import machinery for peroxisomal matrix proteins and other PMPs. These results demonstrate that the Pex11pβ-mediated growth and division of peroxisomes occurs by a maturation pathway (Figure 4.2) following sev-eral steps:

1) Pex11pβ-mediated formation of a peroxisomal subdomain at one side of pre-existing peroxisomes;

2) growth or extension of this subdomain resulting in a peroxisomal membrane compartment which contains some PMPs but no (active) import machinery for matrix proteins;

3) segmentation and constriction of the tubular extension, which requires Pex11pβ, hFis1 and maybe other components, but not DLP1;

4) assembly or activation of the import machinery followed by import of PMPs and matrix proteins;

5) final division into spherical peroxisomes by DLP1.

Similar maturation pathways which are commonly initiated by the formation of an early peroxisomal membrane compartment and its stepwise conversion into a mature, me-tabolically active peroxisome compartment have been proposed for peroxisomal growth/division in yeast (Veenhuis et al. 2000) and in some aspects also resemble ER-dependent peroxisome maturation (Titorenko & Rachubinski 2009; van der Zand et al.

2006). In those models, maturation is achieved by selective and stepwise import of cer-tain PMPs, membrane lipids and matrix proteins.

The observations of the present study clearly demonstrate that growth and division of mammalian peroxisomes (at least the process mediated by Pex11p) is more complex than simple division of a pre-existing organelle and per se represents a process of bio-genesis.

Interestingly, it was observed that the tubular membrane compartment apparently has to segment/constrict in order to import other PMPs and matrix proteins. The block ex-erted e.g. by Pex11pβ-YFP appears to interfere with the assembly of a functional

con-Figure 4.2: Peroxisome multi-plication by growth and division follows a multistep maturation pathway

Pex11pβ mediates the formation of a tubular membrane extension emerg-ing from one side of a spherical, mature peroxisome. The membrane extension grows and acquires a distinct set of PMPs, but is negative for matrix proteins. Afterwards, the membrane extension segments and constricts. This step can be inhibited by expression of Pex11pβ-YFP which keeps the peroxisomes in an elon-gated stage and results in TPA for-mation. The constricted peroxisomal tubule imports other PMPs and matrix proteins and is finally divided by hFis1 and DLP1 into several spherical peroxisomes. Silencing of DLP1 inhibits fission resulting in the accumulation of constricted perox-isomal tubules positive for PMPs and matrix proteins (Koch et al. 2004).

striction/division complex, but not with membrane extension. Interestingly, silencing of DLP1, which acts later on during final membrane scission, leads do accumulation of elongated peroxisomes which are still constricted but contain peroxisomal matrix and membrane proteins (Koch et al. 2004). These results suggest that the consecutive steps of the peroxisomal growth and division process are linked to each other, and may be triggered by the assembly of distinct machineries at the peroxisomal membrane. In this regard it is interesting to note that tubular peroxisomes with bulbous domains have also been observed after overexpression of ScPex25p in a S. cerevisiae pex11/pex25/pex27 triple deletion strain. Furthermore, protein import was impaired by the triple deletion and cells were not able to grow on fatty acids (Rottensteiner et al. 2003b).

PMPs display distinct localizations inside the TPAs – at the tubular extension or the globular domains – although the two compartments show membrane continuity. This observation raises the questions which specific mechanisms restrict the mobility of PMPs and inhibit diffusion into the other compartment. This might be mediated by pro-tein oligomerization, but might also involve a specific lipid environment. In Y. lipolytica a role for lipid microdomains in peroxisome maturation has been proposed (Boukh-Viner et al. 2005; Titorenko & Rachubinski 2009), and a similar mechanism might apply here.

Furthermore, caveolin-1, a protein known to be associated with lipid microdomains forming caveolae, was recently shown to be enriched in the peroxisomal membrane of rat hepatocytes (Woudenberg et al. 2010).

It appears that Pex11pβ-YFP is a valuable tool to enrich or accumulate tubular perox-isomal membranes and might therefore be helpful for specific isolation and analysis of these structures in future experiments. Besides restriction of the mobility of existing proteins in the TPA membrane, also a specific targeting or sorting of PMPs to the globu-lar or extended tubuglobu-lar membrane domain must be achieved. Pex11pβ and hFis1 are likely to be directly targeted to (tubular) peroxisomes in a Pex19p-dependent manner (Jones et al. 2004; Rottensteiner et al. 2004; this study). No evidence for localization to the ER has been obtained. The targeting is likely to occur at the correct sub-compartmental site, regulated e.g. by assembly of the import machineries. If the target-ing would occur indirectly via ER-derived pre-peroxisomal vesicular carriers (section 1.2.2) with distinct cargos, the distinct protein localizations would require a specific tar-geting and fusion of these carriers with the respective TPA domains. They must have the ability to distinguish between globular and tubular membrane domains prior to fusion.

Alternatively, subsequent sorting mechanisms within the peroxisomal membranes could exist.

Membrane growth and extension is expected to involve the transfer of lipids to perox-isomes. A so far unanswered question is how growing peroxisomes are supplied with membrane phospholipids. These lipids are synthesized in the ER and they need to be transported in an efficient manner towards peroxisomes. Three potential mechanisms could apply: A) an ER-derived vesicular transfer; B) membrane-membrane interactions with ER subdomains and a protein-based lipid transfer, resembling lipid transfer to mi-tochondria; C) direct luminal interactions which might be of transient nature (Figure 4.3). Although luminal connections between the ER and TPAs have not been observed, even after massive overexpression of secretory proteins (Figure 3.23), existence of those connections cannot be rigorously denied. Direct luminal connections to ER subdomains have been observed in mouse dendritic cells by three-dimensional image reconstruction (Geuze et al. 2003; Tabak et al. 2003). However, a recent report points towards a nonve-sicular ER-to-peroxisome transfer of phospholipids (Raychaudhuri & Prinz 2008), and a close association of peroxisomes and the smooth ER has been frequently observed (Grabenbauer et al. 2000; Novikoff & Shin 1964; Yamamoto & Fahimi 1987; Zaar et al.

1987).

Figure 4.3: Models for lipid transfer from the ER to peroxisomes

(A) Lipids could be transferred to peroxisomes (Po) from the ER via vesicular carriers. (B) Lipid transfer could occur via membrane-membrane interactions. (C) Direct (transient?) luminal connections could mediate lipid transfer.

Remarkably, the formation of new spherical peroxisomes is inhibited in COS-7 cells con-taining TPAs (Figure 3.13). We have demonstrated that this is due to a block in the divi-sion of pre-existing peroxisomes. However, recent studies in yeast and mammalian cells have shown that peroxisomes can multiply either by division or by de novo formation (Hoepfner et al. 2005; Kim et al. 2006; Motley & Hettema 2007; Nagotu et al. 2008b).

Whereas in yeast peroxisomes only form de novo in the absence of pre-existing perox-isomes and multiply by division in wild-type cells (Motley et al. 2008; Nagotu et al.

2008b), de novo formation and multiplication by division has been proposed to occur simultaneously in mammalian cells (Kim et al. 2006). As the formation of new spherical peroxisomes was not observe here, we might face a situation similar to yeast, where only the complete loss of peroxisomes triggers de novo formation. Alternatively, Pex11pβ might be required for de novo formation as well suggesting an overlap in the components involved. Recent data by Kim et al. (2006) suggested that de novo formation is the major pathway for peroxisome multiplication. The study was based on the as-sumption that all daughter organelles formed by division of pre-existing peroxisomes contain components of their mother peroxisome. However, in the present work it is demonstrated that (Pex11pβ-mediated) peroxisome growth and division is a maturation pathway which includes the import of new proteins into the forming organelles (Figure 3.22). The existence of condition-specific symmetric divisions of peroxisomes can not be excluded. Very recently, Huybrechts et al. (2009) have shown that peroxisome fission is a non-symmetric event, which was similarly observed in early studies of peroxisome proliferation in the regenerating rat liver (Yamamoto & Fahimi 1987). In H. polymorpha non-symmetric peroxisome division appears to be the general mechanism (Nagotu et al.

2008b).