Tail-anchored proteins contain a single transmembrane domain (TMD) followed by a short C-terminal domain extending into the organellar lumen. Tail-anchored proteins are thought to target to the correct subcellular compartment by virtue of general physicochemical properties of their C-termini; however, the machineries that enable correct sorting remain largely elusive. Here we analyzed targeting of the human peroxisomal tail-anchored protein PEX26. Its C-terminal-targeting signal contains two binding sites for PEX19, the import receptor for several peroxisomal membrane proteins. One PEX19-binding site overlapped with the TMD, the other was contained within the luminal domain. Although the PEX19-binding site containing the TMD targeted to peroxisomes to some extent, the luminal site proved essential for correct targeting of the full-length protein, as it prevented PEX26 from mislocalization to mitochondria. Its function as a targeting motif was proved by its ability to insert a heterologous TMD-containing fragment into the peroxisomal membrane. Finally we show that PEX19 is essential for PEX26 import. Analysis of the yeast tail-anchored protein Pex15p revealed that it also harbors a luminal PEX19-binding site that acts as a peroxisomal-targeting motif. We conclude that C-terminal PEX19-binding sites mark tail-anchored proteins for delivery to peroxisomes.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

For human adrenoleukodystrophy protein (ALDP) and its close homologues, a conserved motif has been described recently (11). These proteins constitute half-transporters of the ABC transporter family and are involved in the ATP-dependent transport of yet to be determined metabolites across the peroxisomal membrane. Mutations in the ALDP gene are the cause for X-linked adrenoleukodystrophy, the most common peroxisomal disease (12).To act as a functional mPTS, the basic motif described above critically depends on one or more transmembrane domains (TMDs) in its vicinity so as to insert the PMPs into the membrane (4, 5, 13-16). For a small group of PMPs, particularly PEX3, sorting does not depend on this type of mPTS (16) indicating that these PMPs might reach their final destination in the peroxisomal membrane via a different pathway that could even involve the endoplasmic reticulum (17-19).The largely cytosolic PEX19 is required for the topogenesis of PMPs and is supposed to escort PMPs from the cytosol to the peroxisomal membrane. PEX19 interacts with all PMPs tested so far (7, 20 -23), and its down-regulation by small interfering RNA reduces import of newly synthesized PMPs but not that of matrix proteins (16). Our recent characterization of PEX19-binding sites (BS) in several PMPs of Saccharomyces cerevisiae revealed that these sites have in common a motif of about 15 amino acids and constitute an integral part of the mPTS of yeast PMPs (15). Also human PEX19 was shown to bind to targeting-competent regions of several human PMPs (6, 16); however, whether human PEX19 actually binds the targeting motif, the TMD, or yet another region within the mPTS remained elusive. Furthermore, the two human PEX19-BS that have been characterized in some detail, namely those of PEX13 and PMP34, failed to reveal any obvious similarity (7, 16).Here we predicted PEX19-BS in human PMPs with an algorithm developed for yeast PMPs. The identified PEX19-BS of ALDP was characterized and demonstrated to coincide with its targeting motif. Both the binding of PEX19 and the targeting of the mPTS of ALDP functioned in yeast. In addition, most of the predicted sites were proven to represent true PEX19-BS indicating that human PMPs harbor common PEX19-BS. We conclude that PMP targeting is conserved from yeast to man because of the recognition of the PEX19-BS by the orthologous PEX19 proteins. EXPERIMENTAL PROCEDURESStrains and Media-Escherichia coli strain DH5␣ was used for all plasmid amplifications and isolations. E. coli strains BL21(DE3)

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.

Pex3p is a central component of the import machinery for peroxisomal membrane proteins (PMPs) that can reach peroxisomes via the endoplasmic reticulum (ER) and even trigger de novo peroxisome formation from the ER. Pex19p is the import receptor for type I PMPs, whereas targeting of type II PMPs, of which Pex3p so far represents the only species, does not require Pex19p. Pex3p possesses two domains with distinct function: a short N-terminal domain, which harbors the information for peroxisomal (and ER) targeting, and a C-terminal domain, which faces the cytosol and serves as a docking site for Pex19p, thereby delivering newly synthesized PMPs to the peroxisome. Here we show that the N-terminal domain of Pex3p can be functionally replaced by the N-terminal peroxisomal membrane targeting signal (mPTS) of Pex22p, a supposedly unrelated component of the import machinery for peroxisomal matrix proteins. An exchange of the mPTS of Pex22p by that of Pex3p likewise fully preserved the function of Pex22p. Neither of the two mPTS interacted with Pex19p, and in the absence of Pex19p, colocalization of Pex3p and Pex22p was observed, indicating that also Pex22p is targeted to peroxisomes by a type II mPTS. When a type I mPTS was hooked to the C-terminal domains of Pex22p and Pex3p, function was retained in the case of Pex22p and in part even for Pex3p. The C-terminal domain of Pex3p thus contains the relevant information required for de novo peroxisome formation, thereby challenging the concept of the N terminus of Pex3p being key in that process.Peroxisome biogenesis requires a set of proteins called peroxins, most of which accomplish the faithful import of matrix proteins (1-3). Two types of targeting signals are known to direct proteins to the peroxisomal lumen: a C-terminal peroxisomal targeting signal type 1 (PTS1) 2 comprising the C-terminal tripeptide SKL and conservative variants thereof, and an N-terminal PTS2. The import of peroxisomal membrane proteins (PMPs) follows a distinct route and depends on Pex3p and Pex19p in all of the species analyzed (4) and on Pex16p in mammals (5, 6).The majority of PMPs are recognized by Pex19p during or early after synthesis in the cytosol. Pex19p binding not only protects these hydrophobic proteins from aggregation but also delivers them to the peroxisomal membrane (7-10). Docking of the cargo-loaded Pex19p receptor at the peroxisomal membrane occurs via the C-terminal cytoplasmic domain of Pex3p (10 -13). In a subsequent, poorly understood step, PMPs are released from Pex19p for insertion into the peroxisomal membrane. Although some controversy exists in the literature on the nature of a peroxisomal membrane protein targeting signal (mPTS), it requires one or more transmembrane spans and a targeting-specific sequence that is identical to the Pex19p-binding site in at least a subset of PMPs (4, 10, 11).In mutants with a specific block in matrix protein import, PMPs are normally imported into so-called peroxisomal remnants. In contrast, in the absence of the peroxins required for PM...