RESUMO
Mutations that cause loss of function of GlcNAc-1-phosphotransferase (PTase) lead to the lysosomal storage disorder mucolipidosis II. PTase is the key enzyme of the mannose 6-phosphate (M6P) targeting system that is responsible for tagging lysosomal hydrolases with the M6P moiety for their delivery to the lysosome. We had previously generated a truncated hyperactive form of PTase termed S1S3 which was shown to notably increase the phosphorylation level of secreted lysosomal enzymes and enhance their uptake by cells. Here, we report the 3.4 Å cryo-EM structure of soluble S1S3 lacking both transmembrane domains and cytosolic tails. The structure reveals a high degree of conservation of the catalytic core to full-length PTase. In this dimeric structure, the EF hand of one protomer is observed interacting with the conserved region 4 of the other. In addition, we present a high-quality EM 3D map of the UDP-GlcNAc bound form of the full-length soluble protein showing the key molecular interactions between the nucleotide sugar donor and side chain amino acids of the protein. Finally, although the domain organization of S1S3 is very similar to that of the Drosophila melanogaster (fruit fly) PTase homolog, we establish that the latter does not act on lysosomal hydrolases.
RESUMO
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 Ï-(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.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Glucosiltransferases/metabolismo , Complexo de Golgi/enzimologia , Motivos de Aminoácidos , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/genética , Complexo I de Proteína do Envoltório/metabolismo , Glucosiltransferases/genética , Complexo de Golgi/genética , Células HEK293 , Células HeLa , Humanos , Mucolipidoses/enzimologia , Mucolipidoses/genética , Mutação de Sentido Incorreto , Domínios ProteicosRESUMO
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 ß 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 αß (MLIII αß). 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 αß. 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 αß, 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.
Assuntos
Mutação de Sentido Incorreto , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Retículo Endoplasmático , Complexo de Golgi , Células HEK293 , Células HeLa , Humanos , Mucolipidoses/genética , FenótipoRESUMO
The Golgi enzyme UDP-GlcNAc:lysosomal enzymeN-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase), an α2ß2γ2hexamer, 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.
Assuntos
Lisossomos/enzimologia , Manosefosfatos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Sequência de Aminoácidos , Deleção de Genes , Células HeLa , Humanos , Lisossomos/metabolismo , Dados de Sequência Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Receptores Notch/química , Receptores Notch/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Adaptadoras de Transporte Vesicular/química , Motivos de Aminoácidos , Animais , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Simulação de Acoplamento Molecular , Ligação Proteica , Transporte Proteico , Rede trans-Golgi/metabolismoRESUMO
GABAergic interneuron deficits have been implicated in the epileptogenesis of multiple neurological diseases. While epileptic seizures are a key clinical hallmark of CLN2 disease, a childhood-onset neurodegenerative lysosomal storage disorder caused by a deficiency of tripeptidyl peptidase 1 (TPP1), the etiology of these seizures remains elusive. Given that Cln2 R207X/R207X mice display fatal spontaneous seizures and an early loss of several cortical interneuron populations, we hypothesized that those two events might be causally related. To address this hypothesis, we first generated an inducible transgenic mouse expressing lysosomal membrane-tethered TPP1 (TPP1LAMP1) on the Cln2 R207X/R207X genetic background to study the cell-autonomous effects of cell-type-specific TPP1 deficiency. We crossed the TPP1LAMP1 mice with Vgat-Cre mice to introduce interneuron-specific TPP1 deficiency. Vgat-Cre ; TPP1LAMP1 mice displayed storage material accumulation in several interneuron populations both in cortex and striatum, and increased susceptibility to die after PTZ-induced seizures. Secondly, to test the role of GABAergic interneuron activity in seizure progression, we selectively activated these cells in Cln2 R207X/R207X mice using Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) in in Vgat-Cre : Cln2 R207X/R207X mice. EEG monitoring revealed that DREADD-mediated activation of interneurons via chronic deschloroclozapine administration accelerated the onset of spontaneous seizures and seizure-associated death in Vgat-Cre : Cln2 R207X/R207X mice, suggesting that modulating interneuron activity can exert influence over epileptiform abnormalities in CLN2 disease. Taken together, these results provide new mechanistic insights into the underlying etiology of seizures and premature death that characterize CLN2 disease.
RESUMO
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 cells. However, the relevance and modulatory mechanism of this bidirectional trafficking are unclear. Here, using structure-function analyses, we show that S incorporation into virus-like particles (VLP) and VLP 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.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/metabolismo , Complexo de Golgi/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Vertebrates use the mannose 6-phosphate (M6P)-recognition system to deliver lysosomal hydrolases to lysosomes. Key to this pathway is N-acetylglucosamine (GlcNAc)-1-phosphotransferase (PTase) that selectively adds GlcNAc-phosphate (P) to mannose residues of hydrolases. Human PTase is an α2ß2γ2 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 the identification of a hockey stick-like motif that controls 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 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.
Assuntos
Hidrolases , Manose , Humanos , Hidrolases/metabolismo , Lisossomos/metabolismo , Manose/metabolismo , Fosfatos/metabolismo , Fosforilação , Fosfotransferases/metabolismo , PolissacarídeosRESUMO
While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.
Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Antígenos CD19/metabolismo , Linfócitos B , Glicosilação , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos TRESUMO
Disruption of the mannose 6-phosphate (M-6-P) pathway in HeLa cells by inactivation of the GNPTAB gene, which encodes the α/ß 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.
Assuntos
Catepsina D/genética , RNA Mensageiro/genética , Catepsina D/metabolismo , Células HeLa , Humanos , Manosefosfatos/metabolismo , RNA Mensageiro/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
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 COPß' 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.
Assuntos
Substituição de Aminoácidos , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Glicosilação , Complexo de Golgi , Células HEK293 , Células HeLa , Histidina/genética , Humanos , Lisina/genética , Domínios Proteicos , Proteólise , Glicoproteína da Espícula de Coronavírus/genética , Internalização do VírusRESUMO
The Golgi-localized, gamma-ear containing, ADP-ribosylation factor-binding proteins (GGAs 1, 2, and 3) are multidomain proteins that bind mannose 6-phosphate receptors (MPRs) at the Golgi and play a role, along with adaptor protein complex 1 (AP-1), in the sorting of newly synthesized lysosomal hydrolases to the endolysosomal system. However, the relative importance of the two types of coat proteins in this process is still unclear. Here, we report that inactivation of all three GGA genes in HeLa cells decreased the sorting efficiency of cathepsin D from 97% to 73% relative to wild-type, with marked redistribution of the cation-independent MPR from peripheral punctae to the trans-Golgi network. In comparison, GNPTAB-/- HeLa cells with complete inactivation of the mannose 6-phosphate pathway sorted only 20% of the cathepsin D. We conclude that the residual sorting of cathepsin D in the GGA triple-knockout cells is mediated by AP-1.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Catepsina D/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Lisossomos/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Rede trans-Golgi/metabolismoRESUMO
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.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Aminoácidos Acídicos/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Leucina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sinais Direcionadores de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Citosol/metabolismo , Endocitose/fisiologia , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
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.
Assuntos
Complexo 1 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/química , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Dipeptídeos/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Técnicas In Vitro , Camundongos , Complexos Multiproteicos/química , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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/ß precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/ß 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 ß-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.
RESUMO
The UDP-GlcNAc:lysosomal enzyme, N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-PT), is an α2 ß2 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/ß 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.
Assuntos
Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Dictyostelium/enzimologia , Glicoproteínas/metabolismo , Células HeLa , Humanos , Lisossomos/metabolismo , Proteínas Mutantes/metabolismo , Fosforilação , Domínios Proteicos , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Genes Letais , Alelos , Animais , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , FenótipoRESUMO
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.
Assuntos
Clatrina/metabolismo , Endonucleases/metabolismo , Proteínas de Membrana/metabolismo , Edição de RNA , Transativadores/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Regulação Alostérica , Animais , Sequência de Bases , Membrana Celular/metabolismo , Clatrina/ultraestrutura , Sequência Conservada , Endocitose , Proteínas de Ligação a Ácido Graxo , Loci Gênicos , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Peptídeos/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Filogenia , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Crescimento e Desenvolvimento/genética , Animais , Animais Recém-Nascidos , Células Cultivadas , Embrião de Mamíferos , Feminino , Humanos , Masculino , Mamíferos/embriologia , Mamíferos/genética , Mamíferos/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica/fisiologia , Especificidade por Substrato/genéticaRESUMO
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.