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Vertebrates utilize the mannose 6-phosphate (M6P) recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is GlcNAc-1-phosphotransferase (PTase) that selectively adds GlcNAc-P to mannose residues of the hydrolases. Human PTase is an α2β2γ2 heterohexamer with a catalytic core and several peripheral domains that recognize and bind substrates. Here we report a cryo-EM structure of the catalytic core of human PTase and identification of a hockey-stick-like motif that controls the activation of the enzyme. Movement of this motif out of the catalytic pocket is associated with a rearrangement of part of the peripheral domains that unblocks hydrolase glycan access to the catalytic site, thereby activating PTase. We propose that PTase fluctuates between inactive and active states in solution, and the selective substrate binding of a lysosomal hydrolase through its protein binding determinant to PTase locks the enzyme in the active state to permit glycan phosphorylation. This mechanism would help ensure that only N-linked glycans of lysosomal enzymes are phosphorylated.

The spike (S) protein of SARS-CoV-2 is delivered to the virion assembly site in the ER-Golgi Intermediate Compartment (ERGIC) from both the ER and cis-Golgi in infected cells1-3. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virions and viral fusogenicity are determined by coatomer-dependent S delivery from the cis-Golgi and restricted by S-coatomer dissociation. Although S mimicry of the host coatomer-binding dibasic motif ensures retrograde trafficking to the ERGIC, avoidance of the host-like C-terminal acidic residue is critical for S-coatomer dissociation and therefore incorporation into virions or export for cell-cell fusion. Because this C-terminal residue is the key determinant of SARS-CoV-2 assembly and fusogenicity, our work provides a framework for the export of S protein encoded in genetic vaccines for surface display and immune activation.

Disruption of the mannose 6‐phosphate (M‐6‐P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/β subunits of GlcNAc‐1‐phosphotransferase, results in missorting of newly synthesized lysosomal acid hydrolases to the cell culture media instead of transport to the endolysosomal system. We previously demonstrated that the majority of the lysosomal aspartyl protease, cathepsin D, is secreted in these GNPTAB −/− HeLa cells. However, the intracellular content of cathepsin D in these cells was still greater than that of WT HeLa cells which retained most of the protease, indicating a marked elevation of cathepsin D expression in response to abrogation of the M‐6‐P pathway. Here, we demonstrate that HeLa cells lacking GlcNAc‐1‐phosphotransferase show a fivefold increase in cathepsin D mRNA expression over control cells, accounting for the increase in cathepsin D at the protein level. Further, we show that this increase at the mRNA level occurs independent of the transcription factors TFEB and TFE3. The intracellular cathepsin D can still be trafficked to lysosomes in the absence of the M‐6‐P pathway, but fails to undergo proteolytic processing into the fully mature heavy and light chains. Uptake experiments performed by feeding GNPTAB −/− HeLa cells with various phosphorylated cathepsins reveal that only cathepsin B is capable of partially restoring cleavage, providing evidence for a role for cathepsin B in the proteolytic processing of cathepsin D., Cathepsin D mRNA expression is increased fivefold in HeLa cells when the Man‐6‐P pathway is inactivated. This increase is independent of both TFEB and TFE3. These cells also lack cathepsin B, which is partly responsible for the failure of cathepsin D processing to occur. The effect is reversed by feeding the knockout HeLa cells with cathepsin B.

The SARS‐CoV‐2 spike glycoprotein (spike) mediates viral entry by binding ACE2 receptors on host cell surfaces. Spike glycan processing and cleavage, which occur in the Golgi network, are important for fusion at the plasma membrane, promoting both virion infectivity and cell‐to‐cell viral spreading. We show that a KxHxx motif in the cytosolic tail of spike weakly binds the COPβ’ subunit of COPI coatomer, which facilitates some recycling of spike within the Golgi, while releasing the remainder to the cell surface. Although histidine (KxHxx) has been proposed to be equivalent to lysine within di‐lysine endoplasmic reticulum (ER) retrieval sequences, we show that histidine‐to‐lysine substitution (KxKxx) retains spike at the ER and prevents glycan processing, protease cleavage, and transport to the plasma membrane.

Transport of newly synthesized lysosomal enzymes to the lysosome requires tagging of these enzymes with the mannose 6-phosphate moiety by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), encoded by two genes, GNPTAB and GNPTG. GNPTAB encodes the α and β subunits, which are initially synthesized as a single precursor that is cleaved by Site-1 protease in the Golgi. Mutations in this gene cause the lysosomal storage disorders mucolipidosis II (MLII) and mucolipidosis III αβ (MLIII αβ). Two recent studies have reported the first patient mutations within the N-terminal transmembrane domain (TMD) of the α subunit of GlcNAc-1-phosphotransferase that cause either MLII or MLIII αβ. Here, we demonstrate that two of the MLII missense mutations, c.80T>A (p.Val27Asp) and c.83T>A (p.Val28Asp), prevent the cotranslational insertion of the nascent GlcNAc-1-phosphotransferase polypeptide chain into the endoplasmic reticulum. The remaining four mutations, one of which is associated with MLII, c.100G>C (p.Ala34Pro), and the other three with MLIII αβ, c.70T>G (p.Phe24Val), c.77G>A (p.Gly26Asp), and c.107A>C (p.Glu36Pro), impair retention of the catalytically active enzyme in the Golgi with concomitant mistargeting to endosomes/lysosomes. Our results uncover the basis for the disease phenotypes of these patient mutations and establish the N-terminal TMD of GlcNAc-1-phosphotransferase as an important determinant of Golgi localization.

The glycosyltransferases of the mammalian Golgi complex must recycle between the stacked cisternae of that organelle to maintain their proper steady-state localization. This trafficking is mediated by COPI-coated vesicles, but how the glycosyltransferases are incorporated into these transport vesicles is poorly understood. Here we show that the N-terminal cytoplasmic tails (N-tails) of a number of cis Golgi glycosyltransferases which share a ϕ-(K/R)-X-L-X-(K/R) sequence bind directly to the δ- and ζ-subunits of COPI. Mutations of this N-tail motif impair binding to the COPI subunits, leading to mislocalization of the transferases to lysosomes. The physiological importance of these interactions is illustrated by mucolipidosis III patients with missense mutations in the N-tail of GlcNAc-1-phosphotransferase that cause the transferase to be rapidly degraded in lysosomes. These studies establish that direct binding of the N-tails of mammalian cis Golgi glycosyltransferases with COPI subunits is essential for recycling within the Golgi.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) have revolutionized the management of relapsed and refractory B cell malignancies. Despite high initial response rates, many patients with acute lymphoblastic leukemia (ALL) ultimately relapse after CART19. In contrast, most patients with non-Hodgkin lymphoma experience only partial or no responses. Collectively, To identify pathways responsible for enabling tumor-intrinsic resistance to CART19 we performed a genome-wide loss-of-function screen in the Nalm6 ALL cell line. The second-most enriched gene in this screen was SPPL3 (Figure 1a), encoding a Golgi-resident aspartyl protease. Previous studies have determined that SPPL3 functions to broadly limit protein glycosylation by cleaving glycosyltransferases from the Golgi membrane, impairing their ability to add complex glycans to proteins as they pass through the Golgi (Voss M. et al. EMBO, 2014). Using targeted genomic disruption, we confirmed that loss of SPPL3 results in resistance to CART19 in human ALL and non-Hodgkin lymphoma models (Figures 1b-c). CART19 cells exposed to SPPL3KO ALL demonstrated significantly lower expression of CD69, PD1, Tim3 and CD107a, as well as less activation of the central T cell transcription factors NFAT and NFκB, indicating a global suppression of T cell stimulation. Consistent with its known function, loss of SPPL3 resulted in increased addition of complex glycans to CD19. Surface staining of SPPL3KO cells revealed that CD19 antibodies were less capable of binding this hyperglycosylated CD19. This included decreased binding of the antibody used to construct the anti-CD19 CAR (clone FMC63). Protein modeling revealed that an asparagine residue known to be normally glycosylated on CD19 (N125) is in close physical proximity to the FMC63 binding site (Figure 1d), suggesting that the addition of complex glycans at this site may be responsible for disruption of CAR binding that led to impaired T cell activation. We next turned our attention to CD22, another B cell antigen that is normally glycosylated and the target of CAR therapy. In contrast to CD19, loss of SPPL3 had no impact on CD22 glycosylation or antibody binding. Similarly, loss of SPPL3 did not enable resistance to CD22-targeted CAR T cells. These findings substantiated our hypothesis loss of SPPL3 lead to CART19 failure directly via modifying CD19 glycosylation, and not through another CD19-independent mechanism. To further validate the impact of CD19 glycosylation in regulating CART19 efficacy, we over-expressed SPPL3 in ALL cells, previously shown to promote global hypoglycosylation. We confirmed decreased glycosylation of CD19 (Figure 1e), and found that this resulted in loss of FMC63 binding to CD19 and complete resistance to CART19 activity (Figure 1f). In summary, our findings identify that changes to CD19 glycosylation, either enhanced or decreased, impair the ability of CARs to bind and initiate T cell effector function against malignant B cells. Further, these data identify post-translational protein modification as a novel mechanism of antigen escape from CAR-based T cell immunotherapy. Figure 1 Figure 1. Disclosures Ruella: AbClon: Consultancy, Research Funding; viTToria biotherapeutics: Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; Tmunity: Patents & Royalties. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

The functional redundancy of the three mammalian Golgi-localized, γ-ear–containing, ADP-ribosylation factor-binding proteins (GGAs) was addressed in a previous study. Using insertional mutagenesis, we found that Gga1 or Gga3 homozygous knockout mice were for the most part normal, whereas mice homozygous for two different Gga2 gene-trap alleles exhibited either embryonic or neonatal lethality in the C57BL/6 background, depending on the source of the vector utilized (Byg vs. Tigm, respectively). We now show that the Byg strain harbors a disrupted Gga2 allele that is hypomorphic, indicating that the Byg lethality is attributable to a mechanism independent of GGA2. This is in contrast to the Tigm Gga2 allele, which is a true knockout and establishes a role for GGA2 during the neonatal period. Placement of the Tigm Gga2 allele into the C57BL6/Ola129Sv mixed background results in a lower incidence of neonatal lethality, showing the importance of genetic background in determining the requirement for GGA2 during this period. The Gga2−/− mice that survive have reduced body weight at birth and this runted phenotype is maintained through adulthood.

The UDP-GlcNAc:lysosomal enzyme, N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT), is an α2 β2 γ2 hexamer that mediates the initial step in the formation of the mannose 6-phosphate targeting signal on newly synthesized lysosomal acid hydrolases. The GNPTAB gene encodes the 1256 amino acid long α/β precursor which is normally cleaved at K928 in the early Golgi by Site-1 protease (S1P). Here, we show that removal of the so-called 'spacer-1' domain (residues 86-322) results in cleavage almost exclusively at a second S1P consensus sequence located upstream of K928. In addition, GlcNAc-1-PT lacking spacer-1 exhibits enhanced phosphorylation of several non-lysosomal glycoproteins, while the phosphorylation of lysosomal acid hydrolases is not altered. In view of these effects on the maturation and function of GlcNAc-1-PT, we suggest renaming `spacer-1' the `regulatory-1' domain.

The Golgi enzyme UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2β2γ2 hexamer, mediates the initial step in the addition of the mannose 6-phosphate targeting signal on newly synthesized lysosomal enzymes. This tag serves to direct the lysosomal enzymes to lysosomes. A key property of GlcNAc-1-phosphotransferase is its unique ability to distinguish the 60 or so lysosomal enzymes from the numerous non-lysosomal glycoproteins with identical Asn-linked glycans. In this study, we demonstrate that the two Notch repeat modules and the DNA methyltransferase-associated protein interaction domain of the α subunit are key components of this recognition process. Importantly, different combinations of these domains are involved in binding to individual lysosomal enzymes. This study also identifies the γ-binding site on the α subunit and demonstrates that in the majority of instances the mannose 6-phosphate receptor homology domain of the γ subunit is required for optimal phosphorylation. These findings serve to explain how GlcNAc-1-phosphotransferase recognizes a large number of proteins that lack a common structural motif.

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.

The AP-2 clathrin adaptor differs fundamentally from the related AP-1, AP-3, and AP-4 sorting complexes because membrane deposition does not depend directly on an Arf family GTPase. Instead phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) appears to act as the principal compartmental cue for AP-2 placement at the plasma membrane as well as for the docking of numerous other important clathrin coat components at the nascent bud site. This PtdIns(4,5)P(2) dependence makes type I phosphatidylinositol 4-phosphate 5-kinases (PIPKIs) lynchpin enzymes in the assembly of clathrin-coated structures at the cell surface. PIPKIgamma is the chief 5-kinase at nerve terminals, and here we show that the 26-amino acid, alternatively spliced C terminus of PIPKIgamma661 is an intrinsically unstructured polypeptide that binds directly to the sandwich subdomain of the AP-2 beta2 subunit appendage. An aromatic side chain-based, extended interaction motif that also includes the two bulky C-terminal residues of the short PIPKIgamma635 variant is necessary for beta2 appendage engagement. The clathrin heavy chain accesses the same contact surface on the AP-2 beta2 appendage, but because of additional clathrin binding sites located within the unstructured hinge segment of the beta2 subunit, clathrin binds the beta2 chain with a higher apparent affinity than PIPKIgamma661. A clathrin-regulated interaction with AP-2 could allow PIPKIgamma661 to be strategically positioned for regional PtdIns(4,5)P(2) generation during clathrin-coated vesicle assembly at the synapse.

The adaptor protein AP-1 is the major coat protein involved in the formation of clathrin-coated vesicles at the trans-Golgi network. The prevailing view is that AP-1 recruitment involves coincident binding to multiple low-affinity sites comprising adenosine diphosphate ribosylation factor 1 (Arf-1)–guanosine triphosphate (GTP), cargo sorting signals, and phosphoinositides. We now show that binding of cargo signal peptides to AP-1 induces a conformational change in its core domain that greatly enhances its interaction with Arf-1–GTP. In addition, we provide evidence for cross talk between the dileucine and tyrosine binding sites within the AP-1 core domain such that binding of a cargo signal to one site facilitates binding to the other site. The stable association of AP-1 with Arf-1–GTP, which is induced by cargo signals, would serve to provide sufficient time for adaptor polymerization and clathrin recruitment while ensuring the packaging of cargo molecules into the forming transport vesicles.

The Golgi-associated gamma-adaptin-related ADP-ribosylation factor-binding proteins (GGAs) are critical components of the transport machinery that mediates the trafficking of the mannose 6-phosphate receptors and associated cargo from the trans-Golgi network to the endosomes. The GGAs colocalize in vivo with the clathrin adaptor protein AP-1 and bind to AP-1 in vitro, suggesting that the two proteins may cooperate in packaging the mannose 6-phosphate receptors into clathrin-coated vesicles at the trans-Golgi network. Here, we demonstrate that the sequence, (382)WNSF(385), in the hinge region of GGA1 mediates its interaction with the AP-1 gamma-ear. The Trp and Phe constitute critical amino acids in this interaction. The binding of Rabaptin5 to the AP-1 gamma-ear, which occurs through a FXXPhi motif, is inhibited by a peptide encoding the GGA1 (382)WNSF(385) sequence. Moreover, mutations in the AP-1 gamma-ear that abolish its interaction with Rabaptin5 also preclude its association with GGA1. These results suggest that the GGA1 WXXF-type and Rabaptin5 FXXPhi-type motifs bind to the same or highly overlapping sites in the AP-1 gamma-ear. This binding is modulated by residues adjacent to the core motifs.

The Golgi-localized, γ-ear–containing, adenosine diphosphate ribosylation factor–binding proteins (GGAs) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) in the Golgi and have an essential role in lysosomal enzyme sorting. Here the GGAs and the coat protein adaptor protein–1 (AP-1) were shown to colocalize in clathrin-coated buds of the trans-Golgi networks of mouse L cells and human HeLa cells. Binding studies revealed a direct interaction between the hinge domains of the GGAs and the γ-ear domain of AP-1. Further, AP-1 contained bound casein kinase–2 that phosphorylated GGA1 and GGA3, thereby causing autoinhibition. This could induce the directed transfer of the MPRs from GGAs to AP-1. MPRs that are defective in binding to GGAs are poorly incorporated into AP-1–containing clathrin-coated vesicles. Thus, the GGAs and AP-1 interact to package MPRs into AP-1–containing coated vesicles.

The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a multidomain family of proteins implicated in protein trafficking between the Golgi and endosomes. Recent evidence has established that the cation-independent (CI) and cation-dependent (CD) mannose 6-phosphate receptors (MPRs) bind specifically to the VHS domains of the GGAs through acidic cluster-dileucine motifs at the carboxyl ends of their cytoplasmic tails. However, the CD-MPR binds the VHS domains more weakly than the CI-MPR. Alignment of the C-terminal residues of the two receptors revealed a number of non-conservative differences in the acidic cluster-dileucine motifs and the flanking residues. Mutation of these residues in the CD-MPR cytoplasmic tail to the corresponding residues in the CI-MPR conferred either full binding (H63D mutant), intermediate binding (R60S), or unchanged binding (E56F/S57H) to the GGAs as determined by in vitro glutathione S-transferase pull-down assays. Furthermore, the C-terminal methionine of the CD-MPR, but not the C-terminal valine of the CI-MPR, inhibited GGA binding. Addition of four alanines to the C-terminal valine of the CI-MPR also severely reduced GGA binding, demonstrating the importance of the spacing of the acidic cluster-dileucine motif relative to the C terminus for optimal GGA interaction. Mouse L cells stably expressing CD-MPRs with mutations that enhance GGA binding sorted cathepsin D more efficiently than wild-type CD-MPR. These studies provide an explanation for the observed differences in the relative affinities of the two MPRs for the GGA proteins. Furthermore, they indicate that the GGAs participate in lysosomal enzyme sorting mediated by the CD-MPR.

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001, eLife digest Cells can take proteins and other molecules that are either embedded in, or attached to, their surface membrane and move them inside via a process called endocytosis. This process often involves a protein called clathrin working together with numerous other proteins. Early on, a complex of four proteins, called the adaptor protein-2 complex, interacts with both the ‘cargo’ molecules that are to be taken into the cell, and the cell membrane. Clathrin molecules then assemble into an ordered lattice-like coat, on top of the adaptor protein complex layer. This deforms a small patch of the cell membrane and curves it inwards. The clathrin molecules coat this pocket as it grows in size, until it engulfs the cargo. The pocket quickly pinches off from the membrane to form a bubble-like structure called a vesicle, which is brought into the cell. A family of proteins termed Muniscins were thought to be involved in the early stages of endocytosis and have to arrive at the membrane before the adaptor protein-2 complex and clathrin. But experiments to test this idea—that reduced, or ‘knocked-down’, the production of Muniscins—had given conflicting results. As such, it remained unclear how the small patches of membrane carrying cargo molecules are marked as being destined to become clathrin-coated vesicles. Now Umasankar et al. have studied the role that these proteins play in the early stages of endocytosis in human cells grown in a laboratory. A gene-editing approach was used to precisely disrupt a gene that codes for a Muniscin protein called FCHO2. Umasankar et al. observed that these ‘edited’ cells formed clathrin coats that were more irregular compared with those that form in normal cells. Nevertheless, clathrin-mediated vesicles still formed when this protein was absent, though the process of endocytosis was slower. Similar results were seen when Umasankar et al. used the same approach to disrupt the gene for a related protein called FCHO1 in the same cells. A short fragment of the Muniscin proteins, called the linker, was shown to bind to, and activate, the adaptor protein-2 complex. The linker then recruits this complex to the specific regions of the cell membrane where clathrin-coated vesicles will form. Several dozen other proteins also accumulate where clathrin pockets form; as such, one of the next challenges will be to investigate if this mechanism of locally activating the cargo-gathering machinery is common in living cells. DOI: http://dx.doi.org/10.7554/eLife.04137.002

The expression of mutants with deletions in the N-terminal signal-anchor sequence of cytochrome P450 2C2 and His-tag fusions was examined in Escherichia coli to determine the influence of N-terminal sequences on expression of the protein. Two mutants predicted to be translocated across the membrane inhibited bacterial growth. In other mutants, deletion of the N-terminal transmembrane domain (residues 2-20) reduced expression of functional P450 by about 75% and further deletion of the following linker sequence (residues 21-27) resulted in a modest further decrease. Expression of the mutant with residues 2-27 deleted contrasts with the lack of expression of functional protein if only the linker was deleted, which suggests that the linker sequence is critical for expression only if the protein is inserted into the membrane by the transmembrane domain. Fusion proteins of green fluorescent protein with full-length P450 2C2 and 2C2(Delta2-20) were predominantly membrane-associated in vivo as determined by fluorescence microscopy. Subcellular fractionation of bacteria expressing these proteins and extraction of the proteins from the membrane by high salt or alkaline buffer demonstrated that P450 2C2 was an integral membrane protein while 2C2(Delta2-20) was a peripheral membrane protein that associated with the membrane mainly by hydrophobic interactions. Residues 1-27 of P450 2C2 fused to green fluorescent protein resulted in a redistribution of fluorescence from cytosol to membrane, which, with the deletion studies, indicates that the P450 signal-anchor is both necessary and sufficient for normal membrane targeting and is the sole transmembrane domain of cytochrome P450 2C2 in bacteria. Addition of a His-tag at the N-terminus completely restored wild-type expression levels to the 2C2(Delta2-20) mutants in bacteria. In insect cells, functional 2C2(Delta2-20) was not expressed but an N-terminal His-tag also restored full expression. The increase in expression may be related to decreased association with the membrane mediated by the His-tag.

The GGA family of clathrin adaptor proteins mediates the intracellular trafficking of transmembrane proteins by interacting with DXXLL-type sorting signals on the latter. These signals were originally identified at the carboxy-termini of the transmembrane cargo proteins. Subsequent studies, however, showed that internal DXXLL sorting motifs occur within the N- or C-terminal cytoplasmic domains of cargo molecules. The GGAs themselves also contain internal DXXLL motifs that serve to auto-regulate GGA function. A recent study challenged the notion that internal DXXLL signals are competent for binding to GGAs. Since the question of whether GGA adaptors interact with internal DXXLL motifs is fundamental to the identification of bona fide GGA cargo, and to an accurate understanding of GGA regulation within cells, we have extended our previous findings. We now present additional evidence confirming that GGAs do interact with internal DXXLL motifs. We also summarize the recent reports from other laboratories documenting internal GGA binding motifs.

The GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Here, the VHS domain of GGA2 was shown to bind to the acidic cluster–dileucine motif in the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CI-MPR). Receptors with mutations in this motif were defective in lysosomal enzyme sorting. The hinge domain of GGA2 bound clathrin, suggesting that GGA2 could be a link between cargo molecules and clathrin-coated vesicle assembly. Thus, GGA2 binding to the CI-MPR is important for lysosomal enzyme targeting.

The Golgi-localized, gamma-ear-containing, ADP ribosylation factor-binding family of monomeric clathrin adaptors (GGAs) is known to bind cargo molecules through short C-terminal peptide motifs conforming to the sequence DXXLL (X = any amino acid), while the heterotetrameric adaptors AP-1 and AP-2 utilize a similar but discrete sorting motif of the sequence [D,E]XXXL[L,I]. While it has been established that a single cargo molecule may contain either or both types of these acidic cluster-dileucine (AC-LL) sorting signals, there are no examples of cargo with overlapping GGA and AP-1/AP-2-binding motifs. In this study, we report that the cytosolic tail of low-density lipoprotein receptor-related protein (LRP)9 contains a bifunctional GGA and AP-1/AP-2-binding motif at its carboxy-terminus (EDEPLL). We further demonstrate that the internal EDEVLL sequence of LRP9 also binds to GGAs in addition to AP-2. Either AC-LL motif of LRP9 is functional in endocytosis. These findings represent the first study characterizing the trafficking of LRP9 and also have implications for the identification of additional GGA cargo molecules.

The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxx phi and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxx phi motifs bind to the mu subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the mu or beta subunits of these adaptors as well as the gamma/sigma1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the gamma/sigma1 or alpha/sigma2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the beta1/mu1 and beta2/mu2 hemicomplexes, and the individual beta or mu subunits, was extremely weak or undetectable. The gamma/sigma1 and alpha/sigma2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position -4 compromised binding to the gamma/sigma1 hemicomplex, whereas minimally affecting binding to alpha/sigma2. There was an excellent correlation between binding to the alpha/sigma2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals.

Microarray analysis has identified 89 estrogen target genes. The cis-regulatory elements found upstream of those genes are not well conserved in mouse and human., Background Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. Results Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. Conclusions Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.

The GGAs ( G olgi-localizing, γ -adaptin ear homology domain, A RF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS ( V PS-27, H rs, and S TAM) domain followed by a GAT ( G GA a nd T OM1) domain, a connecting hinge segment, and a C-terminal GAE ( γ - a daptin e ar) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.

Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E. coli and insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.

In cytochrome P450 2C2, the region which links the N-terminal signal anchor with the catalytic domain contains a highly conserved proline-rich region with the sequence, 30-PPGPTPFP-37. Mutation of proline-30 or proline-33 diminished activities of the mutants expressed in COS-1 cells (Chen, C., and Kemper, B. (1996)J. Biol. Chem.271, 28697–28611). Substitution of alanine, proline, or arginine for glycine-32 abolished laurate hydroxylase activity of the proteins expressed in COS-1 cells, which suggests that this residue is also functionally important. To determine the basis for the decreased activity in COS-1 cells, the activities and spectral properties of mutant proteins expressed in insect cells and bacteria were determined. Substitution of alanine for either proline-30 or -33 resulted in reduced expression in insect cells of functional cytochrome P450 hemoprotein and an increase in the expression of inactive cytochrome P420. In contrast, substitution of alanine for proline-31, -35, or -37 resulted in hemoproteins with spectra similar to cytochrome P450 2C2 so that the amount of cytochrome P450 expressed in insect cells correlated with the activities of the mutants in COS-1 cells. The laurate hydroxylase activities per nanomole of cytochrome P450 in insect microsomes were similar for wild type and all mutants, indicating that, once folded, the catalytic activity of membrane-bound cytochrome P450 was not affected by the mutations. Expression in bacteria resulted in diminished expression of cytochrome P450 for all mutants, with the greatest decrease for the proline-30 and -33 mutants, and increased cytochrome P420. In contrast to the insect cell studies, the proline-30 and -33 mutants were inactive, while the other mutants had specific activities 30–70% of cytochrome P450 2C2. These data are consistent with a role for the proline-rich region in efficient assembly of cytochrome P450 2C2 in eukaryotic cells. Mutations of this region also may affect the conformational integrity of the proteins, which was revealed by assays of solubilized bacterially expressed proteins.

Cytochromes P450 (P450) are anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side. Within the peptide sequence linking these two domains in P450 2C2 is a glycine-rich region from residues 22 to 28. To examine the role of this region, deletion and substitution mutations were constructed, and the activities and spectral properties were determined for the mutant proteins expressed in COS-1 cells, insect cells, and bacteria. Deletion of residues 22 to 28 or substitution of 7 valines for this region inactivated the proteins in COS-1 cells, and no P450 species was detected for these mutations in bacteria or insect cells. Substitution of the three glycine residues with alanine or proline or the entire sequence from 22 to 28 with 7 alanines did not reduce lauric acid hydroxylase activity of the proteins expressed in COS-1 cells. Reducing the number of alanines substituted to 4, 3, and 2 progressively decreased activity in COS-1 cells to undetectable levels when 2 alanines were substituted. The loss of activity in COS-1 cells correlated with decreased expression of hemoprotein with a reduced difference spectrum of 450 nm (P450 species) and a corresponding increase in the inactive P420 species in insect cells and bacteria. The activities expressed per nanomole of P450 in insect microsomes were similar for P450 2C2 and the alanine substitution mutants, including the mutant with 2 alanines which was inactive in COS-1 cells. The rates of conversion of P450 to P420 resulting from incubation at 48 degrees C in vitro were not changed sufficiently to explain the increase in expressed P420 observed for the mutants with 3 or 7 alanines substituted. These data are consistent with a role for the residue 22-28 region as a linker that facilitates the folding of P450; however, once the protein is properly folded into the functional P450 species, this region has little influence on the stability and activity of the enzyme.

Microsomal cytochrome P450 is inserted into the membrane of the endoplasmic reticulum (ER) by its N-terminal signal/anchor sequence which also functions as an ER retention signal. To analyze further potential retention signals of cytochrome P450, topological domains of cytochrome P450 2C1 or 2C2, epidermal growth factor receptor, a plasma membrane protein, and bacterial alkaline phosphatase, a secreted protein were exchanged. The N-terminal signal/anchor of cytochrome P450 2C1 functioned as an ER retention signal when placed at the N terminus of several reporter proteins but not when fused at the C terminus of the extracellular domain of epidermal growth factor receptor, with or without a heterologous cytoplasmic domain. Chimeric proteins in which the cytoplasmic domain of cytochrome P450 2C2 was substituted for that of epidermal growth factor receptor were retained in the ER indicating that an independent retention signal is present in the cytoplasmic part of cytochrome P450 2C2. These chimeras were enzymatically active which argues against misfolding as the primary cause of retention. The ER retention signal of the cytoplasmic domain could not be localized to a single amino acid segment by deletion analysis. These results show that cytochrome P450 2C2 contains redundant, complex ER retention signals in its cytoplasmic and N-terminal hydrophobic domains and that the function of the N-terminal signal is context-dependent.

Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.