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ATP-driven processes of peroxisomal matrix protein import

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Published/Copyright: December 14, 2016
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Biological Chemistry
From the journal Biological Chemistry Volume 398 Issue 5-6

Abstract

In peroxisomal matrix protein import two processes directly depend on the binding and hydrolysis of ATP, both taking place at the late steps of the peroxisomal import cycle. First, ATP hydrolysis is required to initiate a ubiquitin-transfer cascade to modify the import (co-)receptors. These receptors display a dual localization in the cytosol and at the peroxisomal membrane, whereas only the membrane bound fraction receives the ubiquitin modification. The second ATP-dependent process of the import cycle is carried out by the two AAA+-proteins Pex1p and Pex6p. These ATPases form a heterohexameric complex, which is recruited to the peroxisomal import machinery by the membrane anchor protein Pex15p. The Pex1p/Pex6p complex recognizes the ubiquitinated import receptors, pulls them out of the membrane and releases them into the cytosol. There the deubiquitinated receptors are provided for further rounds of import. ATP binding and hydrolysis are required for Pex1p/Pex6p complex formation and receptor export. In this review, we summarize the current knowledge on the peroxisomal import cascade. In particular, we will focus on the ATP-dependent processes, which are so far best understood in the model organism Saccharomyces cerevisiae.

Keywords: AAA+-ATPase; peroxin; peroxisome biogenesis; peroxisomes; Pex; protein translocation

Introduction

Peroxisomes are small single-membrane-bound organelles, which are nearly ubiquitous in eukaryotic cells. Typical structural characteristics are their spherical shape and their densely packed, sometimes even crystalline matrix protein content. Peroxisomes have a remarkably dynamic adaptability which allows cells to adapt to different nutrient or stress conditions. The main conserved functions of peroxisomes are beta-oxidation of fatty acids as well as the detoxification of reactive oxygen species (ROS) (Wanders and Waterham, 2006; Wanders, 2014). While the β-oxidation is distributed among mitochondria and peroxisomes in mammals, it is exclusively located in peroxisomes in plants and some lower eukaryotes like the yeast Saccharomyces cerevisiae (Kunau et al., 1988). In addition to these central tasks, peroxisomes are involved in many organism-specific processes like biosynthesis of bile acid and plasmalogens in mammals (Wanders and Waterham, 2006), penicillin biosynthesis in filamentous fungi (Meijer et al., 2010), or photorespiration in plants (Reumann and Weber, 2006).

With such versatile functions illustrating the broad range of physiological contribution of this vital organelle, the wide phenotypic range of peroxisomal disorders comes as no surprise. The two general types of peroxisomal disorders are peroxisomal single enzyme deficiencies (PEDs) and peroxisomal biogenesis disorders (PBDs) (reviewed in Waterham et al., 2016). The latter group, comprising the Zellweger spectrum disorders (ZSD), rhizomelic chondrodysplasia punctata (RCDP) type 1 and 5 as well as peroxisomal fission defects, are caused by mutations of PEX genes, which encode for proteins involved in peroxisomal biogenesis (peroxins).

Although there are a number of highly specialized peroxisomal features and tasks in organisms like methylotrophic yeasts, filamentous yeasts (Woronin bodies), plants (glyoxysomes) or trypanosomes (glycosomes), all peroxisomes share a most widely conserved, elaborate process for the import of matrix and membrane proteins, which form the basis of dynamic peroxisomal function (Pieuchot and Jedd, 2012). In this review, we provide an overview of the matrix protein import process at the peroxisomal membrane with a focus on yeast as model organism. Particularly, we will outline the ATP-dependent steps, namely the ubiquitination of the import receptors and the export of receptors from the membrane back to the cytosol performed by the two AAA+-ATPases (ATPases associated with various cellular activities) Pex1p and Pex6p.

Import of matrix proteins into peroxisomes

Peroxisomes do not contain DNA and all peroxisomal matrix proteins are encoded in the nucleus and synthesized on free ribosomes in the cytosol. The majority of all currently known proteins, which are involved in peroxisomal biogenesis (peroxins), play a role in the import of these proteins into the organelle (Table 1). Strikingly, the matrix proteins are folded prior to import and can traverse the peroxisomal membrane even in larger oligomeric states, highlighted by the import of modified gold particles with a size of 9 nm (Walton et al., 1995; Léon et al., 2006a). In recent years, however, there has been emerging evidence that the import machinery might prefer monomeric substrates (Freitas et al., 2015; Dias et al., 2016). In contrast to later steps, like ubiquitination and export of the receptors, the matrix protein targeting as well as their import into the organellar lumen is ATP-independent (Oliveira et al., 2003; Francisco et al., 2013). This gave rise to the idea of an export-driven import (Schliebs et al., 2010).

Table 1:

Peroxins involved in matrix protein import.

S. cerevisiaeHomo sapiensProperties/function
Matrix protein targeting
 Pex5pPEX5S/PEX5LPTS1 import receptor
 Pex7pPEX7PTS2 import receptor
 Pex9p–Alternative PTS1 import receptor
 Pex18p/Pex21pPEX5LPTS2 co-receptors
Docking and translocation
 Pex8p–Intraperoxisomal, possible cargo release factor
 Pex13pPEX13Docking factor
 Pex14pPEX14Docking factor, import pores
 Pex17p–Associated with the docking complex
Receptor ubiquitination
 Pex2pPEX2E3 enzyme of the RING-finger complex
 Pex4p–E2 enzyme for monoubiquitination
 Pex10pPEX10E3 enzyme of the RING-finger complex
 Pex12pPEX12E3 enzyme of the RING-finger complex
 Pex22p–Membrane anchor of Pex4p
Receptor export
 Pex1pPEX1Receptor dislocation, AAA+-complex
 Pex6pPEX6Receptor dislocation, AAA+-complex
 Pex15pPEX26Membrane anchor of the Pex1p/Pex6p-complex

Most peroxisomal matrix proteins are recognized in the cytosol via specific peroxisomal targeting signals (PTS), located at the extreme C-terminus (PTS1) or near the N-terminus (PTS2). The PTS1 was initially identified as a serine-lysine-leucine (SKL) tripeptide in firefly luciferase (Gould et al., 1987). The definition was later extended to the consensus sequence (S/A/C)-(K/R/H)-L (Gould et al., 1989; Brocard and Hartig, 2006). Furthermore, the importance of preceding amino acid residues for cargo recognition led to a refined definition of the PTS1 as a dodecamer sequence (Brocard and Hartig, 2006). Recent findings suggest that the sequence adjacent to the C-terminal tripeptide provides an independent binding site for the receptor Pex5p, which alone is not sufficient for matrix import and might therefore play a role in ensuring specificity or regulation (Hagen et al., 2015; Styles et al., 2016). Throughout all organisms, the PTS1 pathway is the major import pathway for peroxisomal matrix proteins (Grunau et al., 2009; Reumann et al., 2009; Jung et al., 2010).

In contrast to the PTS1, the PTS2 sequence resides in the N-terminal region of the respective cargo proteins. It was first identified as a cleavable peroxisomal targeting signal in rat thiolase (Swinkels et al., 1991). A consensus sequence for the most common variants of the PTS2, R-(L/V/I/Q)-X2-(L/V/I/H)-(L/S/G/A)-X-(H/Q)-(L/A), was obtained by analysis of known PTS2-containing proteins (Petriv et al., 2004). The crystal structure of a receptor-bound PTS2 peptide revealed the signal sequence to form an α-helix (Pan et al., 2013).

The import of peroxisomal matrix proteins begins with cargo recognition in the cytosol by soluble receptors. In the case of the PTS1 pathway, the main receptor is Pex5p (van der Leij et al., 1993) (Figure 1A). The protein contains several tetratricopeptide repeats (TPRs) in its C-terminal domain, which recognize and bind the PTS1-cargo. The receptor undergoes major conformational changes upon cargo binding, possibly to generate a docking-competent form (Stanley et al., 2006). New results revealed a possible bivalent binding mode with distinct steps for initial cargo recognition and stable formation of the receptor-cargo complex (Hagen et al., 2015). Recently, a novel receptor protein for a subset of PTS1-proteins, which is highly oleate-inducible, has been identified in yeast and was named Pex9p (Effelsberg et al., 2016; Yifrach et al., 2016).

Figure 1: ATP-independent steps of matrix protein import into peroxisomes of Saccharomyces cerevisiae.(A) Import of PTS1-containing matrix proteins. (I) Newly synthesized PTS1-cargo proteins are recognized and bound in the cytosol by the soluble import receptor Pex5p. (II) Pex5p guides the cargo protein to the peroxisomal membrane where the receptor-cargo complex binds to the docking complex (Pex13p, Pex14p, Pex17p). (III) After the formation of a transient pore, composed of at least Pex5p and Pex14p, the PTS1-cargo is translocated across the membrane and released into the peroxisomal lumen. (B) Import of PTS2-containing matrix proteins. (Ia) The soluble receptor Pex7p binds PTS2-cargo proteins in the cytosol. (Ib) Subsequent binding of the co-receptor Pex18p might stabilize the receptor-cargo complex. (II) The ternary receptor-cargo complex binds to the docking complex at the peroxisomal membrane. (III) Pex18p is the pore-forming factor of the PTS2-receptor-cargo complex. It is unclear whether Pex7p is released into the peroxisomal matrix together with its cargo or retained at the membrane.
Figure 1:

ATP-independent steps of matrix protein import into peroxisomes of Saccharomyces cerevisiae.

(A) Import of PTS1-containing matrix proteins. (I) Newly synthesized PTS1-cargo proteins are recognized and bound in the cytosol by the soluble import receptor Pex5p. (II) Pex5p guides the cargo protein to the peroxisomal membrane where the receptor-cargo complex binds to the docking complex (Pex13p, Pex14p, Pex17p). (III) After the formation of a transient pore, composed of at least Pex5p and Pex14p, the PTS1-cargo is translocated across the membrane and released into the peroxisomal lumen. (B) Import of PTS2-containing matrix proteins. (Ia) The soluble receptor Pex7p binds PTS2-cargo proteins in the cytosol. (Ib) Subsequent binding of the co-receptor Pex18p might stabilize the receptor-cargo complex. (II) The ternary receptor-cargo complex binds to the docking complex at the peroxisomal membrane. (III) Pex18p is the pore-forming factor of the PTS2-receptor-cargo complex. It is unclear whether Pex7p is released into the peroxisomal matrix together with its cargo or retained at the membrane.

The PTS2-recepor Pex7p is a member of the WD-40 protein family (Marzioch et al., 1994; Braverman et al., 1997). Unlike Pex5p, Pex7p requires auxiliary factors for the import of proteins into peroxisomes (Figure 1B). While in plants and mammals, this co-receptor function is performed by an isoform of the PTS1-receptor Pex5p (Braverman et al., 1998; Woodward and Bartel, 2005), lower eukaryotes possess additional co-receptors like Pex18p, Pex20p, or Pex21p (Purdue et al., 1998; Titorenko et al., 1998; Sichting et al., 2003; Otzen et al., 2005; Schliebs and Kunau, 2006; Léon and Subramani, 2007). Pex18p and Pex21p were long thought to be redundant, but a new study showed distinct functions for both proteins under different growth conditions (Effelsberg et al., 2015). Human Pex7p was recently reported to form a stable receptor-cargo complex depending on the presence of the co-receptor (Kunze et al., 2015).

After cargo recognition, the cargo-loaded receptors of the PTS1- and the PTS2-pathways are directed to the peroxisomal membrane. This step is facilitated by the docking complex composed of the core components Pex13p and Pex14p in all known species and in some organisms additional proteins, like Pex17p in S. cerevisiae or Pex33p in Neurospora crassa (Brown and Baker, 2008; Managadze et al., 2010). The docking complex is associated with the RING (really interesting new gene)-finger complex, comprising Pex2p, Pex10p and Pex12p. Association of the subcomplexes forms a larger import complex called the importomer, which depends on the intraperoxisomal peripheral membrane protein Pex8p in yeast (Agne et al., 2003). Pex13p is an integral membrane protein with two transmembrane-domains and an intraperoxisomal loop. Both the N-terminus and the C-terminal Src-homology-3(SH3)-domain face the cytosol. Pex13p interacts with a proline-rich segment of Pex14p through its SH3-domain. It also contains an additional intra-peroxisomal Pex14p-binding site (Pires et al., 2003; Schell-Steven et al., 2005). The SH3-domain is also able to bind the PTS1-receptor Pex5p, while the interaction site for the PTS2-receptor Pex7p is located at the N-terminus (Bottger et al., 2000; Stein et al., 2002; Pires et al., 2003). The second main component of the docking complex, Pex14p, is also able to interact with both PTS-receptors (Niederhoff et al., 2005). Pex5p contains several conserved WxxxF/Y motifs within its N-terminal half, which bind to hydrophobic cavities in the N-terminal domain of human Pex14p (Neufeld et al., 2009). For yeast Pex14p, two distinct Pex5p-binding sites in the N-terminal and the C-terminal regions have been described (Niederhoff et al., 2005).

The initial docking of the receptor-cargo complex at the peroxisomal membrane subsequently leads to its incorporation into the translocation machinery. For cargo translocation, several models have been developed throughout the years, with the transient pore model being the one most favored by recent results (Erdmann and Schliebs, 2005). The model is based on the fact that Pex5p has intrinsic lipid-binding affinity and can insert spontaneously into membranes (Kerssen et al., 2006). Moreover, the PTS1-receptor was shown to behave like an integral membrane protein in complex with Pex14p (Gouveia et al., 2000). Furthermore, Pex5p together with Pex14p are sufficient for the import of Pex8p into the peroxisomal matrix (Ma et al., 2009). Finally, the electrophysiological analyses of reconstituted Pex5p-containing complexes isolated from yeast and incubated with a receptor-cargo complex led to the identification of a highly dynamic import pore with a diameter of up to 9 nm, which consists mainly of Pex5p and Pex14p (Meinecke et al., 2010; reviewed in Meinecke et al., 2016) (Figure 1A). This raised the question, whether also PTS2-proteins are imported through a proteinaceous pore and if so, whether both pathways share components. In this respect, it was interesting to note that the yeast PTS2 co-receptor Pex18p was shown to be functionally similar to the N-terminal domain of Pex5p, which mediates lipid- and docking complex-binding (Schäfer et al., 2004). Pex18p was also found in high-molecular membrane complexes together with Pex14p and Pex13p (Grunau et al., 2009). With a similar experimental approach as for the PTS1-pore, the corresponding PTS2-pore, consisting mainly of Pex18p, Pex14p and Pex17p was identified recently (Montilla-Martinez et al., 2015) (Figure 1B). While these data established the existence of distinct pores for the import of peroxisomal PTS1- and PTS2-proteins, our knowledge on the mechanism of cargo translocation is still scarce. In fact, it also is still an open question how the translocated cargo is released into the peroxisomal matrix. In yeast, Pex8p has been implicated in this process, possibly coupled with redox-regulation (Wang et al., 2003a; Ma et al., 2013). This function might be taken over by other members of the translocation machinery in higher eukaryotes, where Pex8p is absent. Indeed, the N-terminus of Pex14p seems to facilitate cargo release in mammals (Freitas et al., 2011).

First ATP-dependent step: ubiquitination of the PTS-receptors

The initial peroxisomal process that requires ATP is the ubiquitination of the import receptors. In general, ubiquitination is a conserved posttranslational protein modification in eukaryotic cells. The attachment of the ubiquitin-moiety to a target protein can affect its binding to partner proteins, subcellular localization or stability (Swatek and Komander, 2016). The ubiquitination of a substrate requires a three-step enzyme cascade. The ubiquitin-activating enzyme (E1) binds ATP and activates ubiquitin via the formation of an adenylated intermediate. Subsequently, the ubiquitin is transferred to the active-site cysteine of a ubiquitin-conjugating enzyme (E2), which cooperates with a ubiquitin-protein ligase (E3). The E3 can bind the Ub-charged E2 enzyme as well as the target protein and contributes to substrate specificity. The E3 can promote the direct transfer of the ubiquitin to the substrate (RING-type E3 family) or generate a thioester-linked Ub-E3 intermediate (HECT-type family and RBR-type family), before it finally attaches the ubiquitin moiety to the target amino acid of the substrate. In some cases, the formed ubiquitin chain can be elongated by RING-like U-box proteins (E4) (Hoppe, 2005; Kerscher et al., 2006; Wenzel et al., 2011; Williamson et al., 2013).

In most cases, the C-terminal glycine 76 of ubiquitin forms a peptide bond to the lysine of the target protein. Less common are examples, where also cysteine, serine or threonine residues are ubiquitinated (Wang et al., 2012).

The attached ubiquitin-signal can be removed from the target protein by deubiquitinating enzymes (DUBs), either in order to complete or to inhibit the ubiquitin-dependent process (Komander et al., 2009).

The functionality of the receptors is governed by their ubiquitination status which contributes to the ATP-dependence of peroxisomal matrix protein import. Ubiquitination of the PTS-receptor module is an essential step in the receptor cycle and depends on the function of the distinct peroxisomal E2- and E3-complexes (Figure 2). This modification primes the receptor for the recognition by the AAA+-type ATPase complex, which functions as a dislocase by pulling the modified receptor from the membrane back to the cytosol, where it is deubiquitinated (Platta et al., 2014, 2016). In case of an impairment of the monoubiquitination-dependent recycling pathway, the PTS-receptors enter a polyubiquitination-dependent pathway, which results in their proteasomal degradation (Platta et al., 2014, 2016) (Figure 2).

Figure 2: ATP-dependent mono- and poly-ubiquitination pathways of Pex5p in S. cerevisiae.Both ubiquitination cascades start with the ATP-dependent activation of a ubiquitin molecule by the soluble E1-enzyme Uba1p in the cytosol. Subsequent steps diverge depending on the pathway. Left: Monoubiquitination of Pex5p. (I) The activated ubiquitin moiety is transferred to the membrane-associated E2-enzyme Pex4p. (II) With the help of two E3-enzymes of the RING-finger complex, Pex12p and Pex10p, Pex5p is ubiquitinated on a conserved cysteine residue. (III) The monoubiquitinated receptor is exported back to the cytosol by the AAA+-complex Pex1p/Pex6p. After deubiquitination by Ubp15p, Pex5p can start a new round of matrix protein import. Right: Polyubiquitination of Pex5p. (I) The main E2-enzyme acting in the polyubiquitination pathway of Pex5p is Ubc4p. (II) Multiple ubiquitin units are transferred to two conserved lysine residues of Pex5p supported by the RING-E3-enzymes Pex2p and Pex10p. (III) Polyubiquitinated Pex5p is exported to the cytosol by the Pex1p/Pex6p complex and subsequently degraded by the 26S proteasome.
Figure 2:

ATP-dependent mono- and poly-ubiquitination pathways of Pex5p in S. cerevisiae.

Both ubiquitination cascades start with the ATP-dependent activation of a ubiquitin molecule by the soluble E1-enzyme Uba1p in the cytosol. Subsequent steps diverge depending on the pathway. Left: Monoubiquitination of Pex5p. (I) The activated ubiquitin moiety is transferred to the membrane-associated E2-enzyme Pex4p. (II) With the help of two E3-enzymes of the RING-finger complex, Pex12p and Pex10p, Pex5p is ubiquitinated on a conserved cysteine residue. (III) The monoubiquitinated receptor is exported back to the cytosol by the AAA+-complex Pex1p/Pex6p. After deubiquitination by Ubp15p, Pex5p can start a new round of matrix protein import. Right: Polyubiquitination of Pex5p. (I) The main E2-enzyme acting in the polyubiquitination pathway of Pex5p is Ubc4p. (II) Multiple ubiquitin units are transferred to two conserved lysine residues of Pex5p supported by the RING-E3-enzymes Pex2p and Pex10p. (III) Polyubiquitinated Pex5p is exported to the cytosol by the Pex1p/Pex6p complex and subsequently degraded by the 26S proteasome.

Monoubiquitination of the PTS-receptors

During the import cascade, the PTS1-receptor Pex5p is monoubiquitinated at a conserved cysteine residue in its N-terminal part. This type of modification is unusual and results not in the formation of the typical amide bond but in a thioester bond between the cysteine of Pex5p and glycine 76 of ubiquitin (Carvalho et al., 2007; Williams et al., 2007; Okumoto et al., 2011).

The ubiquitin-conjugating enzyme that catalyzes this modification is Pex4p (Ubc10p) in S. cerevisiae (Platta et al., 2007; Williams et al., 2007). In 1992, Pex4p was identified as the first E2 enzyme shown to be essential for the biogenesis of an organelle (Wiebel and Kunau, 1992). Work from different yeast species and plants demonstrated that Pex4p is required for both PTS1- and PTS2-dependent matrix protein import (Wiebel and Kunau, 1992; Crane et al., 1994; van der Klei et al., 1998; Zolman et al., 2005). Pex4p associates with peroxisomes via the membrane-protein Pex22p (Koller et al., 1999; Zolman et al., 2005; Williams et al., 2012). The solved crystal structure of S. cerevisiae Pex4p and the soluble part of Pex22p gives important insight into the unique assembly of the peroxisomal E2-complex (Williams et al., 2012, 2013). The N-terminal part of Pex22p is solely required for membrane insertion and can be replaced by the membrane-span of the functionally unrelated Pex3p (El Magraoui et al., 2014). In contrast, the C-terminal part of Pex22p cannot be replaced and harbors the biologic activity of the protein. While Pex4p can genetically be fused to the membrane-portion of Pex3p, it only displays full physiologic activity when the soluble C-terminal part of Pex22p is expressed within the cell (El Magraoui et al., 2014). In summary, Pex22p can be regarded as an activator of the E2 activity of Pex4p (Williams et al., 2012; El Magraoui et al., 2014).

While Pex4p and Pex22p are well conserved in yeasts, fungi and plants, they are seemingly absent in mammals (Kiel et al., 2006). Mammalian cells use the abundant UbcH5a, UbcH5b and UbcH5c to catalyze the monoubiquitination of mammalian Pex5p at the conserved cysteine (Grou et al., 2008). The involved UbcH5-family members also have several other peroxisome-independent cellular target proteins, like IκBα or p53 (Gonen et al., 1999; Saville et al., 2004; Brzovic and Klevit, 2006), which suggests that specificity is provided by the localization.

An intermediate situation seems to be present in the unicellular eukaryote Trypanosoma brucei. Here, Pex4p mediates the ubiquitination of Pex5p (Gualdrón-López et al., 2013). However, the absence of Pex4p only results in a reduced amount of ubiquitinated Pex5p and leads to a mild import defect. Several other ubiquitin-conjugating enzymes are upregulated in this situation, strongly indicating that, similar to mammalian cells, redundant E2 proteins may monoubiquitinate Pex5p in T. brucei (Gualdrón-López et al., 2013).

The monoubiquitination of Pex5p requires the presence of the three conserved RING-domain containing peroxins Pex2p, Pex10p and Pex12p (Kragt et al., 2005; Williams et al., 2008; Platta et al., 2009). Each of them harbors ubiquitin-protein ligase (E3) activity in yeast and plant cells (Williams et al., 2008; Platta et al., 2009; Kaur et al., 2013). They form a distinct subcomplex at the peroxisomal membrane and stabilize each other (Hazra et al., 2002; Agne et al., 2003). In vivo and in vitro studies using truncations of the RING-peroxins indicate a predominant role of Pex12p in the Pex4p-dependent ubiquitination of Pex5p (Platta et al., 2009). The ubiquitination activity of the Pex4p/Pex12p enzyme pair is enhanced by the RING-domain of Pex10p in vitro (El Magraoui et al., 2012). In summary, data from yeast and mammalian cells suggest that the Pex10p/Pex12p unit may represent the active ligase for monoubiquitination of the PTS1-receptor (El Magraoui et al., 2012; Okumoto et al., 2014).

The ubiquitin-moiety is removed prior to a new round of matrix protein import. A mammalian in vitro system demonstrated that the thioester-linked ubiquitin-moiety can be removed non-enzymatically by exposing the modified PTS1-receptor to cytosolic concentrations of glutathione (Grou et al., 2009). Still, most of the Ub-Pex5p conjugate is cleaved enzymatically.

USP9X is the deubiquitinating enzyme acting on monoubiquitinated Pex5p in mammalian cells (Grou et al., 2012). The function of USP9X is not restricted to peroxisomal matrix protein import because it also deubiquitinated TGF-beta (Dupont et al., 2009), the antiapoptotic Mcl1 (Schwickart et al., 2010) or the ubiquitin-ligase MARCH7 (Nathan et al., 2008). Ubp15p is the DUB acting on Pex5p in S. cerevisiae (Debelyy et al., 2011). Ubp15p interacts with the D1-domain of the AAA+-type ATPase Pex6p. In semi-in vitro assays, Ubp15p deubiquitinates the mono- as well as the polyubiquitinated species of Pex5p and its deletion has an impact on peroxisome function under H2O2-stress conditions (Debelyy et al., 2011). The fast removal of the ubiquitin-moiety seems to be important for the optimal progression of the import cycle. This has been shown by the exchange of the conserved Cys at position 6 against a Lys, resulting in the formation of a stable peptide bond to ubiquitin instead of the more labile thioester bond. This leads to a destabilization and proteasomal degradation of Pex5p, which is partially antagonized by Ubp15p (Schwartzkopff et al., 2015).

In general, the current data on Ubp15p as well as the USP9X suggest that redundant DUBs might also be involved in the deubiquitination of Pex5p.

Monoubiquitination also plays an essential role in the PTS2-dependent import pathway. However, the PTS2-import receptor Pex7p does not seem to be ubiquitinated. Instead, the PTS2-co-receptors Pex18p and Pex20p, from S. cerevisiae and Pichia pastoris respectively, are monoubiquitinated on a conserved N-terminal cysteine (Hensel et al., 2011; Liu and Subramani, 2013). In fact, the N-terminal part of these PTS2-co-receptors share similarities with the N-terminal region of Pex5p, including the conserved cysteine. This similarity is also indicated by the fact that the N-terminal region of Pex5p can be functionally replaced by the corresponding portion of Pex18p (Dodt et al., 2001; Schäfer et al., 2004). The monoubiquitination of the PTS2-co-receptor Pex18p seems to be closely linked to the translocation of cargo-bound Pex7p, as mutation of the conserved cysteine in Pex18p arrests Pex7p in a protease-accessible state on the cytosolic side of the membrane (Hensel et al., 2011).

The monoubiquitination of Pex18p is catalyzed by Pex4p in cooperation with the RING-ligases Pex12p and Pex10p (El Magraoui et al., 2013). In line with this, in vivo data suggest that the presence of Pex4p as well as the RING-peroxin complex is required for the monoubiquitination of the PTS2-co-receptor Pex20p in P. pastoris (Liu and Subramani, 2013).

Polyubiquitination of the PTS-receptors

The PTS1-receptor Pex5p can also be polyubiquitinated, which serves as a signal for its degradation by the 26S proteasome. The polyubiquitination is elicited by an impairment of the regular monoubiquitination-dependent recycling pathway, which is the case when constituents of the Pex4p- or AAA+-complex are deleted (Platta et al., 2004; Kiel et al., 2005; Kragt et al., 2005) or when the conserved cysteine of Pex5p is mutated (Williams et al., 2007; Schwartzkopff et al., 2015). Polyubiquitinated Pex5p-species also accumulate in conditional mutants of the 26S proteasome (Platta et al., 2004; Kiel et al., 2005). The Lys48-linked polyubiquitin-chains are attached to two lysine residues in the N-terminal region of Pex5p in S. cerevisiae (Platta et al., 2007; Williams et al., 2007). Formation of the polyubiquitin-chains is mainly catalyzed by the ubiquitin-conjugating enzyme Ubc4p and to a lesser extent by the partial redundant Ubc1p and Ubc5p (Platta et al., 2004; Kiel et al., 2005; Kragt et al., 2005). The Ubc4p-family enzymes are not peroxisome-specific and are known to be involved in several other cellular processes as well (Seufert and Jentsch, 1990; Seufert et al., 1990). The intact peroxisomal RING-ligase complex is required for the polyubiquitination of Pex5p (Platta et al., 2004; Kiel et al., 2005). Based on studies with point mutants and truncations of the RING-peroxins, the E3 enzymes Pex10p (Williams et al., 2008) and Pex2p (Platta et al., 2009) were found to play an important role in the Ubc4p-dependent polyubiquitination of Pex5p. The RING-domain of Pex10p can synergistically enhance the ubiquitination activity of the Ubc4p/Pex2p enzyme pair (El Magraoui et al., 2012).

In contrast to the PTS1-receptor Pex5p, the PTS2-co-receptor Pex18p displays a constitutive turnover in S. cerevisiae (Purdue and Lazarow, 2001; Hensel et al., 2011). This proteasomal degradation is mediated by the polyubiquitination of the first two lysine residues (Hensel et al., 2011). The attachment of the ubiquitin-chains requires the E2 enzyme Ubc4p and the E3 enzymes Pex2p and Pex10p, which act in a partially redundant manner in this context (El Magraoui et al., 2013). The PTS2-co-receptor of P. pastoris, Pex20p is polyubiquitinated on the first conserved lysine (Léon et al., 2006b; Léon and Subramani, 2007; Liu and Subramani, 2013). The question, whether Pex21p is also ubiquitinated in a similar manner, has not yet been addressed.

No evidence for ubiquitination or degradation of Pex7p has been found in S. cerevisiae (Hensel et al., 2011). While Pex18p displays a constitutive breakdown, the Pex7p protein level remains constant (Purdue and Lazarow, 2001; Hensel et al., 2011). In contrast, the P. pastoris PTS2-co-receptor Pex20p is stable and the corresponding Pex7p is polyubquitinated and has a higher turnover rate, especially under methanol-induced conditions, when the PTS2-pathway is not essential (Hagstrom et al., 2014). In Arabidopsis thaliana, endogenous Pex7p is degraded in the proteasome when the dominant-negative GFP-Pex7p is additionally expressed. This process requires the interaction of Pex7p with the small Rab GTPase RabE1c (Cui et al., 2013).

The mechanisms underlying the polyubiquitination of the PTS2-receptors and co-receptors seem to display more species-specific variations than the PTS1-pathway.

In general, the function of the polyubiquitination of the PTS-receptors is the removal of the receptor molecules from the peroxisomal membrane when the regular dislocation pathway is hampered (Platta et al., 2013). This polyubiquitination-pathway is likely to represent a quality control mechanism. It is not essential under normal conditions, because site-directed mutagenesis of the corresponding lysines does not result in a growth defect of S. cerevisiae cells on oleate medium (Platta et al., 2007; Williams et al., 2007). Moreover, polyubiquitination of the PTS-receptors can also be regarded as an alternative export signal. This has been demonstrated by in vitro export assays for S. cerevisiae Pex5p, as a fraction of Pex5p can still be exported in a Pex4p-deficient system, depending on the presence of the two conserved lysine residues (Platta et al., 2007).

A role of the polyubiquitin-chain as the alternative export signal has also been demonstrated for the PTS2-co-receptor Pex20p in P. pastoris cells (Léon and Subramani, 2007). The Pex20p(C8S) mutant can still partially facilitate matrix protein import, as long as the lysine residue required for polyubiquitination of Pex20p is present (Léon and Subramani, 2007).

Therefore, the instability of the PTS-receptors observed in the corresponding mutant cells in most yeast species, plants and certain Zellweger patient cell lines is caused by a rapid degradation via Lys48-linked polyubiquitin chains (Dodt and Gould, 1996; van der Klei et al., 1998; Collins et al., 2000; Zolman and Bartel, 2004; Zolman et al., 2005).

Second ATP-dependent step: formation of the exportomer and export of the PTS-receptors

The modification of the receptors by mono-ubiquitination serves as a signal for their dislocation from the peroxisomal membrane back to the cytosol, where they can start a new round of import (Platta et al., 2007). For yeast as well as human cells, this export step of the PTS1-receptor Pex5p was shown to be ATP-dependent and the AAA+-ATPases Pex1p and Pex6p were identified to be crucially involved in this process (Miyata and Fujiki, 2005; Platta et al., 2005) (Figure 2).

Both Pex1p and Pex6p were originally identified in the 1990s to be important for peroxisomal biogenesis (Erdmann et al., 1991; Voorn-Brouwer et al., 1993). The sequence section around the ATP binding p-loop motif of Pex1p showed a conspicuously high degree of conservation, which was also present in other ATPases like NSF/Sec18p or p97(VCP)/Cdc48p. This indicated that these proteins belong to a novel family of ATPases (Erdmann et al., 1991). Also the later identified Pex6p turned out to belong to this family (Voorn-Brouwer et al., 1993), which were then classified as AAA-ATPases (ATPases associated with various cellular activities) (Kunau et al., 1993), typically comprising at least one conserved 200–230 amino acid spanning AAA-domain. Along with the identification and characterization of further ATPases as well as structural resemblance analyses, this group was widely expanded, new members were included and the family was designated AAA+-family of ATPases (Neuwald et al., 1999). Generally, AAA+-ATPases are characterized by the presence of a AAA+-domain, which structurally is divided into a large α/β- and a small α-helix bundle domain. Typical features of AAA+-domains are conserved motifs like the Walker A and Walker B motifs (Walker et al., 1982), arginine-fingers in the second region of homology (SRH) (Tomoyasu et al., 1993; Karata et al., 1999; Wang et al., 2005; Zhao et al., 2010), the N-linker region (Smith et al., 2004) as well as other charged/polar sensor residues, which all together coordinate ATP with the help of Mg2+ in an extended hydrogen bonding network (reviewed in Wendler et al., 2012). Pex1p and Pex6p together with p97/Cdc48p and NSF/Sec18p belong to the so called class II subfamily of AAA+-ATPases (Iyer et al., 2004; Ammelburg et al., 2006; Erzberger and Berger, 2006), characterized by two consecutively arranged AAA+-domains on one single polypeptide-chain. Typical for some class II AAA+-ATPases like Sec18, Pex1p or Pex6p is that one of these AAA+-domains exhibits a remarkably high conservation and displays the main ATPase activity, whilst the second one reveals higher amino acid variability and often is responsible for nucleotide-dependent complex formation (Whiteheart et al., 1994; Schlee et al., 2001; DeLaBarre and Brunger, 2003; Wang et al., 2003b; Tang et al., 2010; Zhao et al., 2012). In the case of both Pex1p and Pex6p, large (up to 450 amino acids spanning) N-terminal regions are followed by a non-conserved AAA+-domain D1 and a second, highly conserved AAA+-domain D2 (Grimm et al., 2012; Ciniawsky et al., 2015).

Most AAA+-ATPases operate as homo-hexamers. The AAA+-domains congregate into hexameric rings with the nucleotide binding sites located at the interfaces of the protomers (reviewed in Hanson and Whiteheart, 2005; Wendler et al., 2012). Consequently, one hexameric AAA+-ring contains six nucleotide binding sites. In the case of hexamerization of type II AAA+-ATPases, the tandemly arranged AAA+-domains D1 and D2 each form a ring, bringing together 12 nucleotide binding sites in total. Typically, both rings lay on top of each other, forming a so called ‘double-donut’ structure with a central cavity. The arrangement of the AAA+-domains into rings with the nucleotide binding sites connecting the protomers allows an intermolecular sensing of the nucleotide status as well as cooperative movements of the AAA+-domains along the ring, induced by coordinated ATP binding and hydrolysis events (Ogura et al., 2004; Nyquist and Martin, 2014). This principle of a molecular motor enables the chemically fixed energy of ATP to be translated into wide conformational changes and movements, which are transferred by specialized domains of the ATPases and/or bound adaptors to different kind of substrates as indicated by recent EM-snapshots of the type II AAA+-ATPases NSF (Zhao et al., 2015), p97 (Banerjee et al., 2016) and Pex1p/Pex6p (Ciniawsky et al., 2015). Obviously, this concept turned out to be very successful in evolution, since AAA+-family members have been identified across all kingdoms of life, functioning across all compartments and areas of cells (reviewed in Ogura and Wilkinson, 2001). Overall, whenever macromolecules (polypeptides or DNA) have to be remodeled structurally, AAA+-type ATPases seem to be involved (e.g. resolving of protein aggregates, unfolding of proteins for refolding or proteolytic degradation, removing/sorting of soluble or membrane-inserted proteins, unraveling of tight protein-protein complexes or unwinding of DNA double-helices and more). In line with this principle, the translocation of the peroxisomal PTS-receptors from the peroxisomal membrane back to the cytosol is accomplished by the AAA+ motor-proteins Pex1p and Pex6p (reviewed in Grimm et al., 2016a).

Since the vast majority of AAA+-ATPases act as hexameric complexes, Pex1p and Pex6p were expected to function as hexamers, too. Different approaches revealed that Pex1p and Pex6p are able to interact with each other in the presence of ATP or ADP (Faber et al., 1998; Tamura et al., 1998; Kiel et al., 1999). This interaction depends on their nucleotide binding capabilities (Birschmann et al., 2005; Platta et al., 2005; Tamura et al., 2006). Therefore, first presumptions assumed that Pex1p and Pex6p each can form a homo-hexameric complex and that both complexes somehow interact to form a functional dodecameric heteromer. Finally, by using recombinant proteins, the hetero-hexameric nature of the yeast Pex1p/Pex6p complex, composed of three Pex1p and three Pex6p molecules, was demonstrated (Saffian et al., 2012). Additionally, the ATP dependency of its complex formation was clearly confirmed. Thus, Pex1p/Pex6p represents one of the few examples of a hetero-hexameric AAA+-complex, besides the proteasomal Rpt ring (Lander et al., 2012) and the mitochondrial AAA+-proteases (Gerdes et al., 2012) and to our knowledge, it represents the only heteromeric type II AAA+-ATPase.

Recent structural analyses of the ScPex1p/Pex6p complex by electron microscopy techniques revealed an alternating arrangement of Pex1p and Pex6p, forming a characteristic double-ring structure (Blok et al., 2015; Ciniawsky et al., 2015; Gardner et al., 2015) (Figure 3A). These two rings are composed of the D1- and D2-AAA+-domains, and lie on top of each other, surrounding a central cavity. Another unique feature of Pex1p and Pex6p, compared to other type II AAA+-members, are their large N-terminal domains (NTDs). In the case of Pex1p, these domains are situated on top of the assembled Pex1p/Pex6p complex, while in the case of Pex6p the NTDs fold back and flank the D1-D2-double-ring. This arrangement of the Pex6p NTDs results in an atypical triangular shape, when the complex is seen from top (Ciniawsky et al., 2015). In fact the large N-terminal regions of both peroxins, Pex1p as well as Pex6p, are composed of two sequentially arranged domains, each of them resembling a double-ψ-β-barrel fold (Shiozawa et al., 2004; Blok et al., 2015). While the N-terminal domains of Pex6p are known to interact with Pex15p in yeast (Birschmann et al., 2003; Rosenkranz et al., 2006; Grimm et al., 2016b), Pex26p in mammals (Matsumoto et al., 2003a; Nashiro et al., 2011) and AMP9 in plants (Goto et al., 2011) to recruit the Pex1p/Pex6p complex to the peroxisomal membrane, so far no binding partners for the NTDs of Pex1p were identified. However, some binding-capacity of the murine Pex1p NTD to phospholipids was found (Shiozawa et al., 2006), which therefore might support the binding of the Pex1p/Pex6p complex to the peroxisomal membrane. Besides interactions to phospholipids, these double-ψ-β-barrel folds are commonly known to interact with ubiquitin and/or adaptor proteins. The NTD of p97, for example, binds to ubiquitin-chains in concert with its adaptor proteins Ufd1/Npl4 (Ye et al., 2003). Both p97 NTD and Ufd1 exhibit ubiquitin binding activity by their double-ψ-β-barrel folds (Meyer et al., 2000, 2002; Park et al., 2005; Davies et al., 2008). Together, p97 and Ufd1 are involved in the late steps of ERAD (endoplasmic reticulum associated degradation), in which misfolded proteins of the ER are recognized and extracted for proteasomal degradation (Bagola et al., 2011). Interestingly, ERAD and the peroxisomal import machinery share a common principle of function: The respective substrate becomes ubiquitinated and afterwards is extracted from the membrane by type II AAA+-ATPases (Schliebs et al., 2010). In ERAD, misfolded proteins are the substrates for p97 extraction, whereas Pex1p/Pex6p pulls the ubiquitinated PTS-receptors out of the peroxisomal membrane. Although it is unknown how the Pex1p/Pex6p complex contacts its substrates, it seems very likely, that the NTDs are responsible for binding the ubiquitin moiety of the receptors (Figure 3B). As for p97, further components might be involved in this binding process. However, it is speculated that Pex1p and Pex6p with their duplicated double-ψ-β-barrel folds, might bring their own co-factors for ubiquitin binding (Blok et al., 2015; Tan et al., 2016). Other candidates, which could directly or indirectly be involved in binding of the ubiquitinated receptors, are the aforementioned deubiquitinating enzyme Ubp15p in yeast (Debelyy et al., 2011), or AWP1 in mammalian cells (Miyata et al., 2012). AWP1 from rat liver cells was found to interact with Pex6p (surprisingly not in complex with Pex1p) as well as with mono-ubiquitinated Pex5p and actively supported the export process of the PTS1-receptors within an in vitro export assay (Miyata et al., 2012). The mechanism of how Pex1p/Pex6p recognizes and processes its substrates at the peroxisomal membrane is unsolved. However, the recently clarified structural organization of the Pex1p/Pex6p-complex and especially the structural analysis of the complex in different nucleotide states, gave first insights into the molecular movement during the ATP-hydrolysis cycle (Ciniawsky et al., 2015; Tan et al., 2016). In combination with activity measurements of Walker B mutated Pex1p and Pex6p variants, a picture emerges in which the D2-ring of the Pex1p/Pex6p complex acts as motor, whereby especially ATP hydrolysis in the D2 domain of Pex6p drives movements of the entire complex. In contrast, the conserved D2-domain of Pex1p as well as the less conserved AAA+-domains of the D1 ring seem to have no or only minor regulative hydrolysis activity (Blok et al., 2015; Ciniawsky et al., 2015; Gardner et al., 2015). However, nucleotide binding of these domains seems to be crucial for stable complex formation. In parts, this constellation resembles the situation of the soluble F1-ATPase hexamer of the F1F0-ATP-Synthase, which is composed of alternately arranged α and β subunits. Similar to Pex1p/Pex6p, ATP hydrolysis mainly occurs in one of these subunits (in the β subunit), while the other one (the α subunit) takes over regulative functions (Walker et al., 1982; Leyva et al., 2003).

Figure 3: Structure of the Pex1p/Pex6p-complex and model of the ATP-dependent export of Pex5p by the AAA+-complex Pex1p/Pex6p in S. cerevisiae.(A) Surface structure of the Pex1p/Pex6p complex in presence of ATPγS as revealed by negative stain electron microscopy. Pex1p (red/pink) and Pex6p (orange/yellow) are alternately arranged in the heterohexameric complex. The N-terminal domains (gray) of Pex1p and Pex6p are located at the top or to the side of the complex, respectively, giving rise to the triangular appearance. The D1- (pink/yellow) and D2-domains (red/orange) associate in a double-ring structure with a central pore. (B) Model of the function of the AAA+-complex Pex1p/Pex6p in the ATP-dependent export of Pex5p. The model shows a side view of the Pex1p/Pex6p complex with the N-terminal domains facing the peroxisomal membrane. (I) The nucleotide-dependent interaction of the N-terminal domains of Pex6p with membrane-bound Pex15p leads to the recruitment of the Pex1p/Pex6p complex to the peroxisomal membrane. (II) Ubiquitinated Pex5p is recognized by the N-terminal domain of Pex1p. Ubp15p is shown associated with the D1-domain of Pex6p. (III) Pex5p is deubiquitinated by Ubp15p and pulled into the central pore of the Pex1p/Pex6p complex. (IV) Removal of one Pex1p/Pex6p dimer from the model complex reveals the actions of the conserved pore loops (green) located in the D2-ring. Movements of these substrate-binding loops triggered by ATP hydrolysis generate the force to pull Pex5p further into the complex pore. The receptor is at least partially unfolded in this process. (V) Further ATP hydrolysis leads to the release of the Pex1p/Pex6p complex together with Pex5p into the cytosol.
Figure 3:

Structure of the Pex1p/Pex6p-complex and model of the ATP-dependent export of Pex5p by the AAA+-complex Pex1p/Pex6p in S. cerevisiae.

(A) Surface structure of the Pex1p/Pex6p complex in presence of ATPγS as revealed by negative stain electron microscopy. Pex1p (red/pink) and Pex6p (orange/yellow) are alternately arranged in the heterohexameric complex. The N-terminal domains (gray) of Pex1p and Pex6p are located at the top or to the side of the complex, respectively, giving rise to the triangular appearance. The D1- (pink/yellow) and D2-domains (red/orange) associate in a double-ring structure with a central pore. (B) Model of the function of the AAA+-complex Pex1p/Pex6p in the ATP-dependent export of Pex5p. The model shows a side view of the Pex1p/Pex6p complex with the N-terminal domains facing the peroxisomal membrane. (I) The nucleotide-dependent interaction of the N-terminal domains of Pex6p with membrane-bound Pex15p leads to the recruitment of the Pex1p/Pex6p complex to the peroxisomal membrane. (II) Ubiquitinated Pex5p is recognized by the N-terminal domain of Pex1p. Ubp15p is shown associated with the D1-domain of Pex6p. (III) Pex5p is deubiquitinated by Ubp15p and pulled into the central pore of the Pex1p/Pex6p complex. (IV) Removal of one Pex1p/Pex6p dimer from the model complex reveals the actions of the conserved pore loops (green) located in the D2-ring. Movements of these substrate-binding loops triggered by ATP hydrolysis generate the force to pull Pex5p further into the complex pore. The receptor is at least partially unfolded in this process. (V) Further ATP hydrolysis leads to the release of the Pex1p/Pex6p complex together with Pex5p into the cytosol.

Although the order of ATP hydrolysis events within the D2 ring needs further clarification, it appears that during its ATPase cycle, the Pex1p/Pex6p complex carries out a kind of pumping motion, with up and down movements of conserved pore loops of the D2 ring and accompanied by distinct, probably regulative contacts to the D1 ring (Ciniawsky et al., 2015; Tan et al., 2016). Similar to other AAA+-motors (Ishikawa et al., 2001; Weibezahn et al., 2004; Hinnerwisch et al., 2005; Martin et al., 2008), Pex1p/Pex6p might use this mechanism to pull out the PTS-receptors from the membrane by threading them into or even through the cavity of its double-ring structure (Figure 3B).

Beside these ATP-hydrolysis-driven molecular movements, further observations suggest a dynamic attachment of the AAA+-ATPases at the peroxisomal membrane, which is also regulated by the nucleotide cycle (Figure 3B). In yeast, the Pex1p/Pex6p complex exhibits a dual localization, with one fraction anchored to the peroxisomal membrane by its interaction to Pex15p and the second fraction located in the cytosol (Faber et al., 1998; Rosenkranz et al., 2006). In fact, deactivation of the ATP-hydrolysis by a Pex6p Walker B mutation results in a stabilized interaction of the Pex1p/Pex6p complex to Pex15p (Birschmann et al., 2003; Grimm et al., 2016b). It comes as no surprise that this loss of function mutation results in an export defect of the PTS1-receptor Pex5p, indicated by its poly-ubiquitination and accumulation at the peroxisomal membrane. Interestingly, in this situation different other components of the import machinery (e.g. Pex14p of the docking- and Pex12p of the RING-finger-complex) show a stabilized association with the AAA+-complex, indicating a dynamic interplay of the receptor export machinery with earlier steps of the matrix protein import cascade (Grimm et al., 2016b). These findings support the idea of an export-driven import, in which cargo-transfer across the membrane barrier is driven by or mechanistically coupled to the ATP-driven motions of the Pex1p/Pex6p complex, possibly by conformational changes transferred to the PTS-receptor, while it is pulled out (Schliebs et al., 2010).

Most of the above-mentioned observations were made in the yeast system. In mammals, however, the hetero-hexameric arrangement of Pex1p and Pex6p is not assigned yet. Furthermore, the recruiting of the AAA+-ATPases to the peroxisomal membrane appears to slightly differ from the yeast proteins. Mammalian Pex1p predominantly is located in the cytosol (Tamura et al., 1998), likely forming a homotrimeric version (Tamura et al., 2006; Fujiki et al., 2012). Mammalian Pex6p on the other hand primarily binds to the peroxisomal membrane by its interaction with Pex26p (Matsumoto et al., 2003a; Tamura et al., 2006; Nashiro et al., 2011), the orthologue of yeast Pex15p (Elgersma et al., 1997; Matsumoto et al., 2003b). Similar to yeast, interactions between mammalian Pex1p and Pex6p as well as to Pex26p depend on accurate nucleotide binding in the different Walker A motifs (Tamura et al., 2006; Fujiki et al., 2012). While the yeast Pex1p/Pex6p complex appears to stay constantly in the hexameric conformation, the mammalian proteins might disassemble during the ATP cycle, leaving Pex6p connected to the peroxisomal membrane, while Pex1p is released into the cytosol. Although not yet directly observed, such a dissociation mechanism could provide a further level of regulation.

Another interesting observation regarding the connection between the export machinery and the docking complex was identified in the mammalian system. Here it was shown that Pex26p is able to directly bind to the docking component Pex14p and that this interaction is weakened in presence of Pex1p/Pex6p in an ATP-dependent manner (Tamura et al., 2014). Again, similar to the yeast system, these observations point to a dynamic mechanism, in which the pore forming constituents are coupled mechanistically to the Pex1p/Pex6p mediated export steps of the PTS-receptors. Nevertheless, knowledge of the exact import mechanism at the peroxisomal membrane is still scarce and it is going to be exciting to get new insights in the future.

Concluding remarks

Matrix protein import into peroxisomes is facilitated by a remarkable machinery, which translocates folded proteins across the peroxisomal membrane through a transient import pore. The energy requirements for this process seem to be solely fulfilled by the ATP-dependent ubiquitination and export of the PTS-(co-)receptors back to the cytosol. A major aspect of future research will be to determine how protein import and receptor export are coupled mechanistically and how the energy generated during the export steps is transferred to the translocation of proteins into the organelle.

The importance of a detailed understanding of these ATP-dependent processes is highlighted by the fact, that mutations in PEX genes encoding for proteins involved in either ubiquitination (PEX2, PEX10, PEX12) or export (PEX1, PEX6, PEX26) of the PTS1-receptor Pex5p are found in almost 95% of patients diagnosed with a Zellweger spectrum disorder (Waterham and Ebberink, 2012). By far the most common mutation, Pex1p G843D, affects the conserved N-linker motif in the second AAA+-domain of Pex1p, which is involved in ATP binding (Smith et al., 2004). The mutation disrupts Pex1p/Pex6p complex formation and leads to a relatively mild clinical phenotype, making it an interesting therapeutic target (Geisbrecht et al., 1998; Preuss et al., 2002; Majewski et al., 2011). While peroxisomal function in affected cells was shown to improve upon treatment with small-molecule chaperones like arginine or the flavonoid acacetin diacetate, there is no effective therapy for affected patients yet (Zhang et al., 2010; Berendse et al., 2013). In addition to their roles in matrix protein import, the aforementioned peroxins have been implicated in other processes as well. The RING-peroxin Pex2p was reported to be involved in ubiquitin-dependent pexophagy (Williams and van der Klei, 2013; Sargent et al., 2016). In plants, Pex10p seems to be important for peroxisome morphology and organellar contacts with chloroplasts (Prestele et al., 2010). Furthermore, the AAA+-proteins Pex1p and Pex6p have been linked to pre-peroxisomal vesicle fusion, suggesting a controversially discussed possible role in de novo biogenesis of peroxisomes (Faber et al., 1998; Titorenko and Rachubinski, 2000; van der Zand et al., 2012). A more detailed structural and functional understanding of all these ATP-driven processes might open up new possibilities for therapeutic approaches in the future.

Acknowledgments

We apologize to all the scientists whose work could not be cited due to space limitations. We are grateful to Wolfgang Girzalsky for critical reading of the manuscript. We thank Prof. Petra Wendler for providing the structural image of Pex1p/Pex6p (Figure 3A). This work was supported by the Deutsche Forschungsgemeinschaft (SFB642 and FOR1905).

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Received: 2016-9-20
Accepted: 2016-12-11
Published Online: 2016-12-14
Published in Print: 2017-5-1

©2017, Daniel P. Schwerter et al., published by De Gruyter.

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: GTP- and ATP-dependent membrane processes
  4. HIGHLIGHT: SFB 642 – GTP- AND ATP-DEPENDENT MEMBRANE PROCESSES
  5. Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs
  6. Structure-based development of PDEδ inhibitors
  7. Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity
  8. Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport
  9. The small GTPases Ras and Rheb studied by multidimensional NMR spectroscopy: structure and function
  10. Rheb in neuronal degeneration, regeneration, and connectivity
  11. ATP-driven processes of peroxisomal matrix protein import
  12. When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli
  13. Interface analysis of small GTP binding protein complexes suggests preferred membrane orientations
  14. From bacteria to chloroplasts: evolution of the chloroplast SRP system
  15. Involvement of the guanine nucleotide exchange factor Vav3 in central nervous system development and plasticity
  16. The class III phosphatidylinositol 3-kinase Vps34 in Saccharomyces cerevisiae
  17. Strategies in relative and absolute quantitative mass spectrometry based proteomics
  18. Kinetic characterization of apoptotic Ras signaling through Nore1-MST1 complex formation
https://doi.org/10.1515/hsz-2016-0293
Keywords for this article
AAA+-ATPase; peroxin; peroxisome biogenesis; peroxisomes; Pex; protein translocation
Creative Commons
BY-NC-ND 3.0

Articles in the same Issue

  1. Frontmatter
  2. Guest Editorial
  3. Highlight: GTP- and ATP-dependent membrane processes
  4. HIGHLIGHT: SFB 642 – GTP- AND ATP-DEPENDENT MEMBRANE PROCESSES
  5. Common mechanisms of catalysis in small and heterotrimeric GTPases and their respective GAPs
  6. Structure-based development of PDEδ inhibitors
  7. Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity
  8. Mechanisms of action of Rab proteins, key regulators of intracellular vesicular transport
  9. The small GTPases Ras and Rheb studied by multidimensional NMR spectroscopy: structure and function
  10. Rheb in neuronal degeneration, regeneration, and connectivity
  11. ATP-driven processes of peroxisomal matrix protein import
  12. When, how and why? Regulated proteolysis by the essential FtsH protease in Escherichia coli
  13. Interface analysis of small GTP binding protein complexes suggests preferred membrane orientations
  14. From bacteria to chloroplasts: evolution of the chloroplast SRP system
  15. Involvement of the guanine nucleotide exchange factor Vav3 in central nervous system development and plasticity
  16. The class III phosphatidylinositol 3-kinase Vps34 in Saccharomyces cerevisiae
  17. Strategies in relative and absolute quantitative mass spectrometry based proteomics
  18. Kinetic characterization of apoptotic Ras signaling through Nore1-MST1 complex formation
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