Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.     

Dual Localization of Squalene Epoxidase, Erg1p, in Yeast Reflects a Relationship between the Endoplasmic Reticulum and Lipid Particles

Squalene epoxidase, encoded by the ERG1 gene in yeast, is a key enzyme of sterol biosynthesis. Analysis of subcellular fractions revealed that squalene epoxidase was present in the microsomal fraction (30,000 × g) and also cofractionated with lipid particles. A dual localization of Erg1p was confirmed by immunofluorescence microscopy. On the basis of the distribution of marker proteins, 62% of cellular Erg1p could be assigned to the endoplasmic reticulum and 38% to lipid particles in late logarithmic-phase cells. In contrast, sterol Δ²⁴-methyltransferase (Erg6p), an enzyme catalyzing a late step in sterol biosynthesis, was found mainly in lipid particles cofractionating with triacylglycerols and steryl esters. The relative distribution of Erg1p between the endoplasmic reticulum and lipid particles changes during growth. Squalene epoxidase (Erg1p) was absent in an erg1 disruptant strain and was induced fivefold in lipid particles and in the endoplasmic reticulum when the ERG1 gene was overexpressed from a multicopy plasmid. The amount of squalene epoxidase in both compartments was also induced approximately fivefold by treatment of yeast cells with terbinafine, an inhibitor of the fungal squalene epoxidase. In contrast to the distribution of the protein, enzymatic activity of squalene epoxidase was only detectable in the endoplasmic reticulum but was absent from isolated lipid particles. When lipid particles of the wild-type strain and microsomes of an erg1 disruptant were mixed, squalene epoxidase activity was partially restored. These findings suggest that factor(s) present in the endoplasmic reticulum are required for squalene epoxidase activity. Close contact between lipid particles and endoplasmic reticulum may be necessary for a concerted action of these two compartments in sterol biosynthesis.     

The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p)

In Saccharomyces cerevisiae, the C-24 sterol methyltransferase (Erg6p) converts zymosterol to fecosterol, an enzymatic step following C-4 demethylation of 4,4-dimethylzymosterol. Our previous study showed that an endoplasmic reticulum (ER) transmembrane protein, Erg28p, functions as a scaffold to tether the C-4 demethylation enzymatic complex (Erg25p-Erg26p-Erg27p) to the ER. To determine whether Erg28p also interacts with other ergosterol biosynthetic proteins, we compared protein levels of Erg3p, Erg6p, Erg7p, Erg11p and Erg25p in three pairs of erg28 and ERG28 strains. In erg28 strains, the Erg6p level in the ER fraction was decreased by about 50% relative to the wild-type strain, while ER protein levels of the four other ergosterol proteins showed no significant differences. Co-immunoprecipitation experiments, using an erg28 strain transformed with the epitope-tagged plasmid pERG28-HA and proteins detected with anti-HA and anti-Erg6p antibodies, indicated that Erg6p and Erg28p reciprocally co-immunoprecipitate. Further, the split ubiquitin yeast membrane two-hybrid system designed to detect protein interactions between membrane bound proteins also indicated an Erg28p-Erg6p interaction when pERG6-Cub was used as the bait and pERG28-NubG was used as the prey. We conclude that Erg28p may not only anchor the C-4 demethylation enzyme complex to the ER but also acts as a protein bridge to the Erg6p enzyme required for the next ergosterol biosynthetic step.     

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.     

Flux of sterol intermediates in a yeast strain deleted of the lanosterol C-14 demethylase Erg11p

Lanosterol C-14 demethylase Erg11p of the yeast Saccharomyces cerevisiae catalyzes the enzymatic step following formation of lanosterol by the lanosterol synthase Erg7p in lipid particles (LP). Localization experiments employing microscopic inspection and cell fractionation revealed that Erg11p in contrast to Erg7p is associated with the endoplasmic reticulum (ER). An erg11Delta mutation in erg3Delta background, which is required to circumvent lethality of the erg11 defect, did not only change the sterol pattern but also the sterol distribution within the cell. Whereas in wild type the plasma membrane was highly enriched in ergosterol and LP harbored large amounts of sterol precursors in the form of steryl esters, sterol intermediates were more or less evenly distributed among organelles of erg11Delta erg3Delta. This distribution is not result of the erg3Delta background, because in the erg3Delta strain the major intermediate formed, ergosta-7,22-dienol, is also highly enriched in the plasma membrane similar to ergosterol in wild type. These results indicate that (i) exit of lanosterol from LP occurs independently of functional Erg11p, (ii) random supply of sterol intermediates to all organelles of erg11Delta erg3Delta appears to compensate for the lack of ergosterol in this mutant, and (iii) preferential sorting of ergosterol in wild type, but also of ergosta-7,22-dienol in erg3Delta, supplies sterol to the plasma membrane.     

Yeast oxidosqualene cyclase (Erg7p) is a major component of lipid particles.

Oxidosqualene cyclase of the yeast encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study (Athenstaedt, K., Zweytick, D., Jandrositz, A, Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448), Erg7p was identified as a component of yeast lipid particles. Here, we present evidence that Erg7p is almost exclusively associated with this compartment as shown by analysis of enzymatic activity, Western blot analysis, and in vivo localization of Erg7p-GFP. Occurrence of oxidosqualene cyclase in other organelles including the endoplasmic reticulum is negligible. In an erg7 deletion strain or in wild-type cells treated with an inhibitor of oxidosqualene cyclase, the substrate of Erg7p, oxidosqualene, accumulated mostly in lipid particles. Storage in lipid particles of this intermediate produced in excess may provide a possibility to exclude this membrane-perturbing component from other organelles. Thus, our data provide evidence that lipid particles are not only a depot for neutral lipids, but also participate in coordinate sterol metabolism and trafficking and serve as a storage site for compounds that may negatively affect membrane integrity.     

A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system

Sterol biosynthesis occurs in the ER and most sterol biosynthetic enzymes have transmembrane domains. However, due to difficulties in characterizing membrane protein-protein interactions, the nature of the sterol biosynthetic complex as well as in vivo interactions between various enzymes have not been described. We employed a split-ubiquitin membrane protein yeast two-hybrid system to characterize interactions between sterol biosynthetic proteins. Fourteen bait constructs were co-transformed into a reporter yeast strain with 14 prey constructs representing all sterol enzymatic reactions beginning with the synthesis of squalene. Our results not only confirmed several previous interactions, but also allowed us to identify novel interactions. Based on these results, ergosterol biosynthetic enzymes display specific protein-protein interactions forming a functional complex we designate, the ergosome. In this complex, Erg11p, Erg25p, Erg27p, and Erg28p appear to form a core center that can interact with other enzymes in the pathway. Also Erg24p and Erg2p, two enzymes that are sensitive to morpholine antifungals, appear to interact with one another; however, the profile of protein interaction partners appears to be unique. Erg2p and Erg3p, two enzymes catalyzing sequential reactions also appear to have different interaction partners. Our results provide a working model as to how sterol biosynthetic enzymes are topologically organized not only in yeast but in plant and animal systems that share many of these biosynthetic reactions.     

Regulated targeting of the monotopic hairpin membrane protein Erg1 requires the GET pathway

The guided entry of tail-anchored proteins (GET) pathway targets C-terminally anchored transmembrane proteins and protects cells from lipotoxicity. Here, we reveal perturbed ergosterol production in ∆get3 cells and demonstrate the sensitivity of GET pathway mutants to the sterol synthesis inhibiting drug terbinafine. Our data uncover a key enzyme of sterol synthesis, the hairpin membrane protein squalene monooxygenase (Erg1), as a non-canonical GET pathway client, thus rationalizing the lipotoxicity phenotypes of GET pathway mutants. Get3 recognizes the hairpin targeting element of Erg1 via its classical client-binding pocket. Intriguingly, we find that the GET pathway is especially important for the acute upregulation of Erg1 induced by low sterol conditions. We further identify several other proteins anchored to the endoplasmic reticulum (ER) membrane exclusively via a hairpin as putative clients of the GET pathway. Our findings emphasize the necessity of dedicated targeting pathways for high-efficiency targeting of particular clients during dynamic cellular adaptation and highlight hairpin proteins as a potential novel class of GET clients.     

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.     

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast

In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.     

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.     

Proteomic analysis of detergent-resistant membranes from Candida albicans

Lipid rafts are membrane microdomains with a higher amount of saturated fatty acids and sterols than the rest of the membrane. They are more resistant to the action of non-anionic detergents, and are called, for this reason, detergent-resistant membranes (DRMs). Lipid rafts are involved in many cellular processes, like signaling, cytokinesis, response to environment, etc., and therefore must contain important proteins. We have obtained a fraction enriched in proteins from Candida albicans DRMs. The sample has been analyzed by SDS-PAGE and 29 proteins have been identified including markers for lipid rafts in Saccharomyces cerevisiae, like Pma1p and a glycosylphosphatidylinositol (GPI)-anchored protein belonging to the Phr family. Ecm33p, a GPI-anchored protein involved in cell wall biogenesis, has been found for the first time in lipid rafts. We have also identified proteins implicated in protein glycosylation, like the mannosyltransferases Mnn7p, Pmt2p and Mnt1p; proteins involved in lipid metabolism, like Erg11p and Scs7p; and heat shock proteins, like Ssa1p and Hsp90p. Most of the proteins identified are located in plasma, mitochondrial, Golgi or ER membranes, supporting the postulated existence of lipid-raft domains in all the membranes.     

Generation of a complete, soluble, and catalytically active sterol 14 alpha-demethylase-reductase complex.

Sterol 14 alpha-demethylation is one of the key steps of sterol biosynthesis in eukaryotes and is catalyzed by cytochrome P450 sterol 14 alpha-demethylase (other names being CYP51 and P45014DM) encoded by ERG11. This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by NCPR1 (NCP1), which is also associated with the endoplasmic reticulum. A diglycine linker recognition site (Gly-Gly-Ile-Glu-Gly-Arg-Gly-Gly) for the protease factor Xa, also containing a thrombin recognition site, was inserted just beyond the N-terminal hydrophobic segment of Candida albicans Erg11p. This modified enzyme was heterologously expressed at a level of 2.5 nmol of Erg11p/mg of protein as an integral endoplasmic reticulum protein. Following purification, treatment of the modified protein with factor Xa or thrombin resulted in sequence-specific cleavage and production of a soluble N-terminal truncated Erg11p which exhibited spectral characteristics identical to those of the purified full-length, wild-type form. Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system. The complex was disrupted by high-salt concentration, reflecting the importance of electrostatic forces in the protein-protein interaction. The results demonstrate the membrane anchor serves to localize Erg11p to the ER where the substrate is located, but is not essential in either Ncpr1p or Erg11p activity. The possibility of cocrystallization of an active soluble eukaryotic 14 alpha-demethylase can be envisaged.     

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.     

Expression of the Cymbidium ringspot virus 33-kilodalton protein in Saccharomyces cerevisiae and molecular dissection of the peroxisomal targeting signal

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.     

A specific structural requirement for ergosterol in long-chain fatty acid synthesis mutants important for maintaining raft domains in yeast

Fungal sphingolipids contain ceramide with a very-long-chain fatty acid (C26). To investigate the physiological significance of the C26-substitution on this lipid, we performed a screen for mutants that are synthetically lethal with ELO3. Elo3p is a component of the ER-associated fatty acid elongase and is required for the final elongation cycle to produce C26 from C22/C24 fatty acids. elo3delta mutant cells thus contain C22/C24- instead of the natural C26-substituted ceramide. We now report that under these conditions, an otherwise nonessential, but also fungal-specific, structural modification of the major sterol of yeast, ergosterol, becomes essential, because mutations in ELO3 are synthetically lethal with mutations in ERG6. Erg6p catalyzes the methylation of carbon atom 24 in the aliphatic side chain of sterol. The lethality of an elo3delta erg6delta double mutant is rescued by supplementation with ergosterol but not with cholesterol, indicating a vital structural requirement for the ergosterol-specific methyl group. To characterize this structural requirement in more detail, we generated a strain that is temperature sensitive for the function of Erg6p in an elo3delta mutant background. Examination of raft association of the GPI-anchored Gas1p and plasma membrane ATPase, Pma1p, in the conditional elo3delta erg6(ts) double mutant, revealed a specific defect of the mutant to maintain raft association of preexisting Pma1p. Interestingly, in an elo3delta mutant at 37 degrees C, newly synthesized Pma1p failed to enter raft domains early in the biosynthetic pathway, and upon arrival at the plasma membrane was rerouted to the vacuole for degradation. These observations indicate that the C26 fatty acid substitution on lipids is important for establishing raft association of Pma1p and stabilizing the protein at the cell surface. Analysis of raft lipids in the conditional mutant strain revealed a selective enrichment of ergosterol in detergent-resistant membrane domains, indicating that specific structural determinants on both sterols and sphingolipids are required for their association into raft domains.     

Conformational plasticity of the lipid transfer protein SCP2

The nonspecific lipid transfer protein sterol carrier protein 2 (SCP2) is involved in organellar fatty acid metabolism. A hydrophobic cavity in the structure of SCP2 accommodates a wide variety of apolar ligands such as cholesterol derivatives or fatty acyl-coenzyme A (CoA) conjugates. The properties of this nonspecific lipid binding pocket are explored using NMR chemical shift perturbations, paramagnetic relaxation enhancement, amide hydrogen exchange, and 15N relaxation measurements. A common binding cavity shared by different physiological ligands is identified. NMR relaxation measurements reveal that residues in the three C-terminal alpha-helices within the lipid binding region exhibit mobility at fast (picosecond to nanosecond) and slow (microsecond to millisecond) time scales. Ligand binding is associated with a considerable loss of peptide backbone mobility. The observed conformational dynamics in SCP2 may play a role for the access of hydrophobic ligands to an occluded binding pocket. The C-terminal peroxisomal targeting signal of SCP2 is specifically recognized by the Pex5p receptor protein, which conducts cargo proteins toward the peroxisomal organelle. Neither the C-terminal targeting signal nor the N-terminal precursor sequence interferes with lipid binding by SCP2. The alpha-helices involved in lipid binding also mediate a secondary interaction interface with the Pex5p receptor. Silencing of conformational dynamics of the peptide backbone in these helices upon either lipid or Pex5p binding might communicate the loading state of the cargo protein to the targeting receptor.