Characterization of the Tautomycin Biosynthetic Gene Cluster from Streptomyces spiroverticillatus Unveiling New Insights into Dialkylmaleic Anhydride and Polyketide Biosynthesis

Tautomycin (TTM) is a highly potent and specific protein phosphatase inhibitor isolated from Streptomyces spiroverticillatus. The biological activity of TTM makes it an important lead for drug discovery, whereas its spiroketal-containing polyketide chain and rare dialkylmaleic anhydride moiety draw attention to novel biosynthetic chemistries responsible for its production. To elucidate the biosynthetic machinery associated with these novel molecular features, the ttm biosynthetic gene cluster from S. spiroverticillatus was isolated and characterized, and its involvement in TTM biosynthesis was confirmed by gene inactivation and complementation experiments. The ttm cluster was localized to a 86-kb DNA region, consisting of 20 open reading frames that encode three modular type I polyketide synthases (TtmHIJ), one type II thioesterase (TtmT), five proteins for methoxymalonyl-S-acyl carrier protein biosynthesis (Ttm-ABCDE), eight proteins for dialkylmaleic anhydride biosynthesis and regulation (TtmKLMNOPRS), as well as two additional regulatory proteins (TtmF and TtmQ) and one tailoring enzyme (TtmG). A model for TTM biosynthesis is proposed based on functional assignments from sequence analysis, which agrees well with previous feeding experiments, and has been further supported by in vivo gene inactivation experiments. These findings set the stage to fully investigate TTM biosynthesis and to biosynthetically engineer new TTM analogs.Tautomycin (TTM)² is a polyketide natural product first isolated in 1987 from Streptomyces spiroverticillatus (1). The structure and stereochemistry of TTM were established on the basis of chemical degradation and spectroscopic evidence (2-4). TTM contains several features not common to polyketide natural products, including a spiroketal group, a methoxymalonate-derived unit, and an acyl chain bearing a dialkylmaleic anhydride moiety. Structurally related to TTM is tautomycetin (TTN), which was first isolated in 1989 from Streptomyces griseochromogenes following the discovery of TTM (5, 6). The structure of TTN was deduced by chemical degradation and spectroscopic analysis (6), and its stereochemistry was established by comparison of spectral data with those of TTN degradation products and synthetic fragments (7). Both TTM and TTN exist as tautomeric mixtures composed of two interconverting anhydride and diacid forms in approximately a 5:4 ratio under neutral conditions (Fig. 1A) (1, 2).Open in a separate windowFIGURE 1.A, structures of TTM and TTN in anhydride or diacid forms, and biosynthetic origin of the dialkylmaleic anhydride by feeding experiments using ¹³C-labeled acetate and propionate. The methoxymalonate-derived unit in TTM is highlighted by the dotted oval. R, polyketide moiety of TTM or TTN. B, selected natural product inhibitors of PP-1 and PP-2A featuring a spiroketal or dialkylmaleric anhydride moiety. C, selected natural products containing a dialkylmaleic anhydride moiety.Early studies of TTM revealed its ability to induce morphological changes in leukemia cells (8). However, it was later realized that TTM is a potent and specific inhibitor of protein phosphatases (PPs) PP-1 and PP-2A (9). PP-1 and PP-2A are two of the four major serine/threonine protein phosphatases that regulate diverse cellular events such as cell division, gene expression, muscle contraction, glycogen metabolism, and neuronal signaling in eukaryotic cells (10-12). Many natural product PP-1 and PP-2A inhibitors are known, including okadaic acid (13), calyculin-A (14), phoslactomycin, spirastrellolide, and cantharidin (15) (Fig. 1B), as well as TTM (16, 17), and TTN (18). They have served as useful tools to study PP-involved intracellular events in vivo and as novel leads for drug discovery (10-12). Among these PP inhibitors, TTM and TTN are unique because of their PP-1 selectivity. Despite their structural similarities, TTM exhibits potent specific inhibition of PP-1 and PP-2A with IC₅₀ values of 22-32 nm and only a slight preference for PP-1 (18). Conversely, TTN shows nearly a 40-fold higher binding affinity to PP-1 (IC₅₀ = 1.6 nm) than to PP-2A (IC₅₀ = 62 nm) (18). Because the major structural differences between TTM and TTN reside in the region distal to the dialkylmaleic anhydride moiety (Fig. 1A), it has been proposed that differences in these moieties might be responsible for the PP-1 selectivity (17-19). Finally, TTN also has an impressive immuno-suppressive activity (20, 21), which is apparently devoid for TTM. Clearly, the structural differences between these two polyketides translate into large, exploitable differences in bio-activities, yet an understanding of the biosynthetic origins of these differences remains elusive.The spiroketal and dialkylmaleic anhydride features of TTM are uncommon for polyketide natural products, as is the methoxymalonate-derived unit (Fig. 1A). Few studies have been carried out for spiroketal biosynthesis, yet it is reasonably common among the phosphatase inhibitors such as calyculin A, okadaic acid, and a few others (Fig. 1B). Less common, but still found in the phosphatase inhibitor cantharidin, as well as TTM and TTN, is the dialkylmaleic anhydride moiety (Fig. 1B); this unit appears in a number of other natural products (Fig. 1C), although the biosynthetic steps leading to this reactive moiety (a protected version of a dicarboxylate) have not been rigorously investigated. Feeding experiments with ¹³C-labeled precursors indicated that the anhydride of TTM and TTN is assembled from a propionate and an as yet undefined C-5 unit (Fig. 1A), which would require novel chemistry for polyketide biosynthesis (22). TTM differentiates itself from all known PP-1 and PP-2A inhibitors by virtue of its unique combination of both the dialkymaleic anhydride and spiroketal functionalities.Multiple total syntheses of TTM and a small number of analogs have been reported, confirming the predicted structure and absolute stereochemistry and facilitating structure-activity relationship studies on PP inhibition and apoptosis induction (19, 23-25). These studies revealed that: (i) the C22-C26 carbon chain and the dialkylmaleic anhydride are the minimum requirements for TTM bioactivity; (ii) the C18-C21 carbon chain and 22-hydroxy group are important for PP inhibition; (iii) the spiroketal moiety determines the affinity to specific protein phosphatases; (iv) the active form is most likely the dicarboxylate; and (v) 3′-epi-TTM exhibits 1,000-fold less activity than TTM. However, taken as a whole, none of the analogs had an improved potency or selectivity for PP-1 inhibition than the natural TTM (19, 22-25). As a result, a more specific inhibitor of PP-1 is urgently awaited to differentiate the physiological roles of PP-1 and PP-2A in vivo and to explore PPs as therapeutic targets for drug discovery.We have undertaken the cloning and characterization of the TTM biosynthetic gene cluster from S. spiroverticillatus as the first step toward engineering TTM biosynthesis for novel analogs (26). We report here: (i) cloning and sequencing of the complete ttm gene cluster, (ii) determination of the ttm gene cluster boundaries, (iii) bioinformatics analysis of the ttm cluster and a proposal for TTM biosynthesis, and (iv) genetic characterization of the TTM pathway to support the proposed pathway. Of particular interest has been the identification of genes possibly related to dialkylmaleic anhydride biosynthesis, the unveiling of the ttm polyketide synthase (PKS) genes predicted to select and incorporate four different starter and extender units for TTM production, and the apparent lack of candidate genes associated with spiroketal formation. These findings now set the stage to engineer TTM analogs for novel PP-1- and PP-2A-specific inhibitors by applying combinatorial biosynthetic methods to the TTM biosynthetic machinery.     

The Mechanism of Antifungal Action of a New Polyene Macrolide Antibiotic Antifungalmycin 702 from Streptomyces padanus JAU4234 on the Rice Sheath Blight Pathogen Rhizoctonia solani

Antifungalmycin 702, a new polyene macrolide antibiotic produced by Streptomyces padanus JAU4234, has a broad antifungal activity and may have potential future agricultural and/or clinical applications. However, the mechanism of antifungal action of antifungalmycin 702 remains unknown. Antifungalmycin 702 strongly inhibited mycelial growth and sclerotia formation/germination of Rhizoctonia solani. When treated with antifungalmycin 702, the hyphae morphology of R . solani became more irregular. The membrane and the cellular organelles were disrupted and there were many vacuoles in the cellular space. The lesion in the plasma membrane was detected through the increase of membrane permeability, lipid peroxidation and leakage of cell constituents. In summary, antifungalmycin 702 may exert its antifungal activity against R . solani by changing the structure of cell membranes and the cytoskeleton and interacting with the organelles.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Purification, Characterization, and Antifungal Activity of Chitinase from Streptomyces venezuelae P10

Streptomyces venezuelae P₁₀ could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N’-diacetylchitobiose.     

Post-PKS Tailoring Steps of a Disaccharide-Containing Polyene NPP in Pseudonocardia autotrophica

A novel polyene compound NPP identified in a rare actinomycetes, Pseudonocardia autotrophica KCTC9441, was shown to contain an aglycone identical to nystatin but to harbor a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine, which led to higher solubility and reduced hemolytic activity. Although the nppDI was proved to be responsible for the transfer of first polyene sugar, mycosamine in NPP biosynthesis, the gene responsible for the second sugar extending glycosyltransferase (GT) as well as NPP post-PKS tailoring mechanism remained unknown. Here, we identified a NPP-specific second sugar extending GT gene named nppY, located at the edge of the NPP biosynthetic gene cluster. Targeted nppY gene deletion and its complementation proved that nppY is indeed responsible for the transfer of second sugar, N-acetyl-glucosamine in NPP biosynthesis. Site-directed mutagenesis on nppY also revealed several amino acid residues critical for NppY GT function. Moreover, a combination of deletions and complementations of two GT genes (nppDI and nppY) and one P450 hydroxylase gene (nppL) involved in the NPP post-PKS biosynthesis revealed that NPP aglycone is sequentially modified by the two different GTs encoded by nppDI and nppY, respectively, followed by the nppL-driven regio-specific hydroxylation at the NPP C10 position. These results set the stage for the biotechnological application of sugar diversification for the biosynthesis of novel polyene compounds in actinomycetes.     

Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

Phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) are encoded by HIS4 in yeast and by hisIE in bacteria and catalyze the second and the third step, respectively, in the histidine biosynthetic pathway. By complementing a hisI mutation of Escherichia coli with an Arabidopsis cDNA library, we isolated an Arabidopsis cDNA (At-IE) that possesses these two enzyme activities. The At-IE cDNA encodes a bifunctional protein of 281 amino acids with a calculated molecular mass of 31,666 D. Genomic DNA-blot analysis with the At-IE cDNA as a probe revealed a single-copy gene in Arabidopsis, and RNA-blot analysis showed that the At-IE gene was expressed ubiquitously throughout development. Sequence comparison suggested that the At-IE protein has an N-terminal extension of about 50 amino acids with the properties of a chloroplast transit peptide. We demonstrated through heterologous expression studies in E. coli that the functional domains for the PRA-CH (hisI) and PRA-PH (hisE) resided in the N-terminal and the C-terminal halves, respectively, of the At-IE protein.     

Antifungal activity of valinomycin, a peptide antibiotic produced by Streptomyces sp. Strain M10 antagonistic to Botrytis cinerea

A strain of Streptomyces sp. (M10) antagonistic to Botrytis cinerea was isolated from orchard soil obtained from Jeju Island, Korea. An antifungal substance (CN1) was purified from the culture extracts of the strain, and then identified as valinomycin through extensive spectroscopic analyses. Valinomycin showed potent in vitro antifungal activity against Botrytis cinerea and also in vivo control efficacy against Botrytis blight development in cucumber plants. Overall, the disease control efficacy of valinomycin was similar to that of vinclozolin, a commercial fungicide. This study provides the first report on the disease control efficacy of valinomycin against Botrytis blight.     

Development of New Modular Genetic Tools for Engineering the Halophilic Archaeon Halobacterium salinarum

Our ability to genetically manipulate living organisms is usually constrained by the efficiency of the genetic tools available for the system of interest. In this report, we present the design, construction and characterization of a set of four new modular vectors, the pHsal series, for engineering Halobacterium salinarum, a model halophilic archaeon widely used in systems biology studies. The pHsal shuttle vectors are organized in four modules: (i) the E. coli’s specific part, containing a ColE1 origin of replication and an ampicillin resistance marker, (ii) the resistance marker and (iii) the replication origin, which are specific to H. salinarum and (iv) the cargo, which will carry a sequence of interest cloned in a multiple cloning site, flanked by universal M13 primers. Each module was constructed using only minimal functional elements that were sequence edited to eliminate redundant restriction sites useful for cloning. This optimization process allowed the construction of vectors with reduced sizes compared to currently available platforms and expanded multiple cloning sites. Additionally, the strong constitutive promoter of the fer2 gene was sequence optimized and incorporated into the platform to allow high-level expression of heterologous genes in H. salinarum. The system also includes a new minimal suicide vector for the generation of knockouts and/or the incorporation of chromosomal tags, as well as a vector for promoter probing using a GFP gene as reporter. This new set of optimized vectors should strongly facilitate the engineering of H. salinarum and similar strategies could be implemented for other archaea.     

Host Generated siRNAs Attenuate Expression of Serine Protease Gene in Myzus persicae

Background

Sap sucking hemipteran aphids damage diverse crop species. Although delivery of ds-RNA or siRNA through microinjection/feeding has been demonstrated, the efficacy of host-mediated delivery of aphid-specific dsRNA in developing aphid resistance has been far from being elucidated.

Methodology/Principal Findings

Transgenic Arabidopsis expressing ds-RNA of Myzus persicae serine protease (MySP) was developed that triggered the generation of corresponding siRNAs amenable for delivery to the feeding aphids. M. persicae when fed on the transgenic plants for different time intervals under controlled growth conditions resulted in a significant attenuation of the expression of MySP and a commensurate decline in gut protease activity. Although the survivability of these aphids was not affected, there was a noticeable decline in their fecundity resulting in a significant reduction in parthenogenetic population.

Conclusions/Significance

The study highlighted the feasibility of developing host based RNAi-mediated resistance against hemipteran pest aphids.     

Identification and Analysis of the Paulomycin Biosynthetic Gene Cluster and Titer Improvement of the Paulomycins in Streptomyces paulus NRRL 8115

The paulomycins are a group of glycosylated compounds featuring a unique paulic acid moiety. To locate their biosynthetic gene clusters, the genomes of two paulomycin producers, Streptomyces paulus NRRL 8115 and Streptomyces sp. YN86, were sequenced. The paulomycin biosynthetic gene clusters were defined by comparative analyses of the two genomes together with the genome of the third paulomycin producer Streptomyces albus J1074. Subsequently, the identity of the paulomycin biosynthetic gene cluster was confirmed by inactivation of two genes involved in biosynthesis of the paulomycose branched chain (pau11) and the ring A moiety (pau18) in Streptomyces paulus NRRL 8115. After determining the gene cluster boundaries, a convergent biosynthetic model was proposed for paulomycin based on the deduced functions of the pau genes. Finally, a paulomycin high-producing strain was constructed by expressing an activator-encoding gene (pau13) in S. paulus, setting the stage for future investigations.     

Deletions Generated by the Transposon Tn10 in the srl recA Region of the ESCHERICHIA COLI K-12 Chromosome

A negative regulatory gene for the srl operon (srlR) was recognized by the characteristics of an insertion mutation generated by the transposon Tn10 determining tetracycline resistance. This finding is discussed in light of previous hypotheses on the regulation of the srl genes, which mediate metabolism of glucitol (i.e., sorbitol). Mapping showed that the order of genes in this region is: srlR srlD srlC recA alaS. Using two different methods, five mutations of both srl and recA were detected. The phenotype conferred by these mutations, UV sensitivity and extreme recombination deficiency, is characteristic of standard recA point mutants. Three of the mutations were deletions that also removed the genes for tetracycline resistance of the nearby transposon. A fourth mutation ended at a distance from Tn10 sufficient to allow separation of the two by recombination following P1 transduction; our tests did not allow us to conclude whether this mutation was an inversion or a deletion. The fifth mutation was a deletion that seemed to end immediately adjacent to the boundary of Tn10, proximal to recA. Mechanisms for the generation of these srl recA mutations are discussed.     

Altered Contractility of Skeletal Muscle in Mice Deficient in Titin’s M-Band Region

We investigated the contractile phenotype of skeletal muscle deficient in exons MEx1 and MEx2 (KO) of the titin M-band by using the cre-lox recombination system and a multidisciplinary physiological approach to study skeletal muscle contractile performance. At a maximal tetanic stimulation frequency, intact KO extensor digitorum longus muscle was able to produce wild-type levels of force. However, at submaximal stimulation frequency, force was reduced in KO mice, giving rise to a rightward shift of the force-frequency curve. This rightward shift of the force-frequency curve could not be explained by altered sarcoplasmic reticulum Ca²⁺ handling, as indicated by analysis of Ca²⁺ transients in intact myofibers and expression of Ca²⁺-handling proteins, but can be explained by the reduced myofilament Ca²⁺ sensitivity of force generation that we found. Western blotting experiments suggested that the excision of titin exons MEx1 and MEx2 did not result in major changes in expression of titin M-band binding proteins or phosphorylation level of the thin-filament regulatory proteins, but rather in a shift toward expression of slow isoforms of the thick-filament-associated protein, myosin binding protein-C. Extraction of myosin binding protein-C from skinned muscle normalized myofilament Ca²⁺ sensitivity of the KO extensor digitorum longus muscle. Thus, our data suggest that the M-band region of titin affects the expression of genes involved in the regulation of skeletal muscle contraction.     

Cyanobactericidal Effect of Streptomyces sp. HJC-D1 on Microcystis auruginosa

An isolated strain Streptomyces sp. HJC-D1 was applied to inhibit the growth of cyanobacterium Microcystis aeruginosa FACHB-905. The effect of Streptomyces sp. HJC-D1 culture broth on the cell integrity and physiological characteristics of M. aeruginosa FACHB-905 was investigated using the flow cytometry (FCM), enzyme activity and transmission electron microscopy (TEM) methods. Results showed that the growth of M. aeruginosa FACHB-905 was significantly inhibited, and the percentage of live cells depended on the culture broth concentration and exposure time. The activities of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) increased with exposure concentration and exposure time, and the significant increase of reactive oxygen species (ROS) led to the disruption of the subcellular structure of M. aeruginosa FACHB-905, and caused the increase of malondialdehyde (MDA). Furthermore, TEM observation suggested the presence of three stages (cell breakage, organelle release and cell death) for the cyanobactericidal process of Streptomyces sp. HJC-D1. Therefore, Streptomyces sp. HJC-D1 not only affected antioxidant enzyme activities and ROS level, but also destroyed the subcellular structure of M. aeruginosa FACHB-905, demonstrating excellent cyanobactericidal properties.     

Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis

Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the β2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Gα_s and Gα_q, localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Gα_s and Gα_q remained associated with the PM and these endosomal membranes, whereas β2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.G protein-coupled receptors (GPCRs)² belong to a superfamily of seven transmembrane-spanning proteins that respond to a diverse array of sensory and chemical stimuli (1–4). Activation of GPCRs through the binding of specific agonists induces conformational changes that allow activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) (5, 6). To ensure that the signals are controlled in magnitude and duration, activated GPCRs are rapidly desensitized through phosphorylation carried out by G protein-coupled receptor kinases (GRKs) (7). This facilitates β-arrestin binding and promotes receptor uncoupling from the G protein (8, 9). In addition to its role in GPCRs desensitization, β-arrestins promote the translocation of the receptor to the endocytic machinery involving clathrin and adaptor protein-2 (AP-2), thereby facilitating receptor removal from the plasma membrane (10–15). Once internalized, some GPCRs may even continue to signal from endosomes (16).Although GPCR internalization is generally considered to be an agonist-dependent phenomenon, some evidence suggests that GPCRs can be endocytosed even in the absence of agonist, a process known as constitutive internalization (17–20). The role of constitutive internalization of GPCRs is not clear. One interesting study on cannabinoid CB1 receptors in neurons has shown that constitutive internalization from the somatodendritic and not axonal membrane is responsible for the overall redistribution of receptors from the somatodentritic to the axonal membrane (17). Another study on the melanocortin MC4 receptor raised the possibility that constitutive endocytosis could be a consequence of the basal activity of the receptor (18).Even less is known about the potential trafficking of the transducer of GPCR signaling, the G protein (21). Generally, the binding of the agonist to the GPCR promotes the exchange of GDP on the Gα protein for GTP and allows the dissociation of the trimeric G protein into Gα-GTP and Gβγ dimer subunits (5, 22). Then, the activated G proteins target different effectors (23, 24). G proteins are localized primarily to the PM where they interact with GPCRs; however, it is not known whether G proteins always remain at the PM or whether they might move into cells along endocytic pathways. Previous work showed that Gα_s does not colocalize with β2 receptor on internal compartments after agonist stimulation, but the cellular distribution of Gα_s was not examined (25).In general, cargo proteins at the plasma membrane (PM) enter the cell through a variety of endocytic mechanisms that can be divided into two main groups: clathrin-dependent endocytosis (CDE) and clathrin-independent endocytosis (CIE). CDE is used by PM proteins such as the transferrin receptor (TfR) that contain specific cytoplasmic sequences recognized by adaptor proteins allowing a rapid and efficient internalization through clathrin-coated vesicles (26, 27). In contrast, CIE is used by PM proteins that lack adaptor protein binding sequences including cargo proteins such as the major histocompatibility complex class I protein (MHCI), the glycosylphosphatidylinositol-anchored protein CD59, and integrins (28–30). In HeLa cells CIE is independent of, and CDE dependent on, clathrin and dynamin and thus the two different endocytic pathways are distinct and well defined (31). After internalization in separate vesicles, MHCI-containing vesicles from CIE and transferrin receptor-containing vesicles from CDE subsequently fuse with the early endosomal compartment that is associated with Rab5 and the early endosomal antigen 1 (EEA1) (32). TfR is recycled back out to the PM in Rab4- and Rab11-dependent processes. In contrast, some MHCI is trafficked on to late endosomes and lysosomes for degradation, and some is recycled back out to the PM along tubular endosomes that lack TfR and emanate from the juxtanuclear area. Recycling of MHCI back to the PM requires the activity of Arf6, Rab22, and Rab11 (33, 34).In this study, we analyzed the trafficking of GPCRs and their G proteins in the presence and absence of agonist in HeLa cells. We examined the trafficking of two prototypical class I GPCRs: the β2 adrenergic receptor (coupled to Gα_s) and the M3 acetylcholine muscarinic receptor (coupled to Gα_q). We find that β2 and M3 receptors traffic constitutively via CIE, and then, in the presence of agonist, they switch to the CDE pathway. We also examined the role of the third intracellular loop of the M3 receptor in this process. To our knowledge, this study represents the most comprehensive analysis of constitutive trafficking of class I GPCRs and related Gα proteins. We demonstrate that GPCRs are versatile PM cargos that utilize different mechanisms of internalization depending upon ligand activation. Considering the high level of homology between class I GPCRs, this evidence could be applicable to the other members of this family.