The observation that mammalian cells deficient in Pex19p lack peroxisome membrane structures points towards a role for this peroxin in PMP biogenesis (24
). We found that Pex19p interacts with Pex3p, Pex10p, Pex11pβ, Pex12p, Pex13p, and Pex16p but not with Pex2p, Pex11pα, Pex14p, PMP22, and PMP24 in the yeast two-hybrid system (Fig. A). Similar results were recently reported by Sacksteder et al. (28
). For unknown reasons, these authors failed to detect the binding of Pex19p to Pex3p and to Pex16p. That Pex19p can indeed interact with Pex3p in the yeast two-hybrid system was also recently reported by Ghaedi et al. (14
). The fact that Pex10p, Pex11pβ, Pex12p, Pex13p, and Pex16p interact with Pex19p in the yeast two-hybrid system suggests that, in this system, other (noninteracting) PMPs with two transmembrane-spanning domains (e.g., Pex2p, Pex11pα, and PMP24) are also theoretically capable of being targeted to the nucleus. However, negative two-hybrid results do not necessarily prove a lack of interaction; for example, Sacksteder et al. (28
) showed that Pex19p binds to PMP34 and Pex14p in blot overlay assays. Yet, the extent of binding is difficult to deduce from the provided data. Why Pex14p interacts with Pex19p in a blot overlay assay (28
) but not in the yeast two-hybrid system (Fig. A) (28
) is currently not clear. The fact that human Pex14p strongly interacts with Pex5p in the yeast two-hybrid system (M. Fransen and P. P. Van Veldhoven, unpublished data) eliminates the possibility of poor expression or failure to be targeted to the nucleus. Other mammalian Pex19p-binding proteins identified by using the yeast two-hybrid system are the ATP binding cassettes half-transporters ALDP, ALDRP, and PMP70 (15
In order to verify whether or not the observed two-hybrid interactions are direct or bridged by an endogenous yeast protein, we further examined the binding properties of Pex19p in vitro. However, we encountered the problem that PMPs containing two or more TMDs were poorly expressed and insoluble; thus, only the Pex3p-Pex19p interaction could be confirmed in vitro. Yet, based on the fact that (i) our tests were performed on S. cerevisiae
cells by using mammalian peroxins and (ii) human Pex19p fails to complement the corresponding yeast deletion mutant (16
), it is likely that the interactions that we observed in the yeast two-hybrid system are direct. In conclusion, our results confirm that human Pex19p can bind multiple integral PMPs and extend the list of PMPs with which Pex19p interacts to include Pex16p.
To determine whether human Pex19p functions as a soluble receptor for the targeting of integral PMPs to the peroxisome, the Pex19p-BDs and the peroxisomal sorting signals of Pex3p, Pex12p, Pex13p, and Pex16p were delineated. Deletion analysis studies demonstrated that, for Pex3p and Pex12p, the Pex19p-BDs and the peroxisomal sorting signals are distinct (Fig. and ). For Pex13p and Pex16p, the domains essential for Pex19p binding were also necessary for protein sorting (Fig. and ). However, further random mutagenesis studies demonstrated that the Pex19p-BD and the peroxisomal sorting signal of Pex13p(145-233)
could be functionally separated (Fig. ). The separate introduction of all the missense mutations in the full-length Pex13p molecule showed that the amino acids from position 175 to 196 are essential for Pex19p binding but not for protein sorting (Fig. ). Although similar experiments were not performed for Pex16p, these results indicate that human Pex19p does not function as a general soluble targeting receptor for integral PMPs. A similar conclusion was obtained for P. pastoris
). However, it has to be noted that the targeting elements of PMP70, ALDP, ALDPR, Pex11pβ, and Pex14p are bound by Pex19p (15
). But, as Sacksteder et al. (28
) pointed out, these results do not indisputably establish that Pex19p binds to the PMP targeting signals in these elements. More specifically, as we report in this study that for Pex13p, the Pex19p-BDs and the peroxisomal targeting elements of these proteins might be functionally separated.
Since random mutagenesis studies revealed that single amino acid substitutions like V178E and L191P affect the peroxisomal localization of Pex13p(145-233)
(Fig. ) but not of the full-length Pex13p molecule (Fig. A), we conducted a refined Pex13p deletion analysis to investigate whether Pex13p contains more than one peroxisomal sorting determinant and/or whether regions flanking the peroxisomal sorting determinant cooperate with the mPTS (Fig. ). We could demonstrate that the central matrix loop of Pex13p alone contains sufficient information to direct a reporter protein to the peroxisome (Fig. A). However, increasing the hydrophobicity of this loop by adding one of the flanking TMDs enhanced the overall sorting efficiency (Fig. A and ). These observations suggest that the mPTS and the TMDs are separable entities that need to coexist for proper Pex13p biogenesis. Importantly, the portion of the central matrix loop of Pex13p (amino acids 155 to 233) that is bound to the peroxisome membrane cannot be removed from this membrane with 0.1 M Na2
, pH 11 (Fig. ). Similar observations have been reported for the mPTSs of CbPMP47 and PpPex3p, and it has been suggested that these mPTSs are tightly anchored to the peroxisomal membrane via another integral PMP (7
). We could also show that, in the presence of flanking sequences, the minimal targeting motif in the central matrix loop of Pex13p can be further narrowed down (Fig. C and D). One explanation may be that these flanking sequences cooperate with the mPTS to enhance its overall sorting efficiency. However, further progressive truncation experiments yielded two nonoverlapping deletion proteins, each displaying a partial peroxisomal staining pattern. This result demonstrates that Pex13p possesses multiple, partially functional mPTSs. A cooperative recognition of these multiple sorting signals may be important for regulating the topology of Pex13p within the peroxisomal membrane.
Since it has been reported that the prenylation status of a protein can affect its binding properties (23
), we investigated the impact of the CaaX farnesylation consensus sequence on the binding properties of Pex19p. We demonstrated that Pex19pΔCaaX has a strongly reduced binding affinity for Pex10p, Pex11pβ, Pex12p, and Pex13p but not for Pex3p and Pex16p (Table ). These results suggest that prenylation of Pex19p is important for its association with Pex10p, Pex11pβ, Pex12p, and Pex13p but not for its association with Pex3p and Pex16p. However, in view of the recently published report that bacterially expressed Pex19p does bind Pex12p and Pex13p in a blot overlay assay (28
), it seems that, under specific experimental conditions, nonfarnesylated Pex19p does display a certain affinity for these peroxins. Currently, we don't know whether these observed differences are the result of the different methodologies employed. In the context of the dilemma of whether or not farnesylation of Pex19p is absolutely required for its function, it is also interesting to mention the reported discrepancy that a C296S mutant of human Pex19p does (28
) or does not (24
) complement peroxisome biogenesis in pex19−/−
fibroblasts. Although our experiments do not solve this dilemma, they demonstrate that the presence of the farnesylation motif of Pex19p strongly enhances its affinity for some PMPs. Similar conclusions were also drawn for S. cerevisiae
). In this organism, the interaction of Pex3p with Pex19p requires farnesylation of the latter molecule (16
). Mapping the PMP-BDs of Pex19p further revealed that the prenylation-dependent interactions require not only the CaaX motif but also the N terminus (Fig. ). This observation may indicate either that these PMPs bind to identical sites of Pex19p or that the deletions change the folding of Pex19p in such a manner that binding to distinct sites is affected. On the other hand, the prenyl-independent interactions are mediated by distinct domains of Pex19p (Fig. ). These results suggest that the prenyl-dependent and the prenyl-independent interactions of Pex19p may serve another function in PMP biogenesis. It is tempting to speculate that Pex19p may bind to Pex3p and Pex16p to form a functional PMP import complex at the peroxisome membrane. What the function of Pex19p in this complex might be is not clear. Snyder et al. (29
) recently suggested that, in P. pastoris
cells, Pex19p might have a chaperone-like role at the peroxisome membrane. Nevertheless, since Pex19p is predominantly present in the cytosol, this molecule most likely also has other biological functions. However, the fact that human Pex19p binds integral PMPs at regions distinct from the mPTS indicates that this peroxin does not function as a general soluble targeting receptor for integral PMPs.