The Yeast Clathrin Adaptor Protein Complex 1 Is Required for the Efficient Retention of a Subset of Late Golgi Membrane Proteins

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

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ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Molecular Biology of the Cell ◽

10.1091/mbc.9.6.1323 ◽

1998 ◽

Vol 9 (6) ◽

pp. 1323-1337 ◽

Cited By ~ 73

Author(s):

Yunxiang Zhu ◽

Linton M. Traub ◽

Stuart Kornfeld

Keyword(s):

Protein Complex ◽

Binding Sites ◽

Adaptor Protein ◽

Limiting Factor ◽

Golgi Membrane ◽

Gtp Hydrolysis ◽

Adp Ribosylation ◽

High Affinity ◽

Golgi Membranes ◽

Adp Ribosylation Factor

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

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Regulation of clathrin adaptor protein complex‐1(AP‐1) by a Laa1 protein containing complex

The FASEB Journal ◽

10.1096/fasebj.2020.34.s1.00639 ◽

2020 ◽

Vol 34 (S1) ◽

pp. 1-1

Author(s):

Mara Colleen Duncan

Keyword(s):

Protein Complex ◽

Adaptor Protein ◽

Clathrin Adaptor

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Exomer: a coat complex for transport of select membrane proteins from the trans-Golgi network to the plasma membrane in yeast

The Journal of Cell Biology ◽

10.1083/jcb.200605106 ◽

2006 ◽

Vol 174 (7) ◽

pp. 973-983 ◽

Cited By ~ 86

Author(s):

Chao-Wen Wang ◽

Susan Hamamoto ◽

Lelio Orci ◽

Randy Schekman

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Cell Surface ◽

Protein Complex ◽

Adaptor Protein ◽

Nucleotide Exchange ◽

Trans Golgi Network ◽

Peripheral Proteins ◽

Golgi Network

Ayeast plasma membrane protein, Chs3p, transits to the mother–bud neck from a reservoir comprising the trans-Golgi network (TGN) and endosomal system. Two TGN/endosomal peripheral proteins, Chs5p and Chs6p, and three Chs6p paralogues form a complex that is required for the TGN to cell surface transport of Chs3p. The role of these peripheral proteins has not been clear, and we now provide evidence that they create a coat complex required for the capture of membrane proteins en route to the cell surface. Sec7p, a Golgi protein required for general membrane traffic and functioning as a nucleotide exchange factor for the guanosine triphosphate (GTP)–binding protein Arf1p, is required to recruit Chs5p to the TGN surface in vivo. Recombinant forms of Chs5p, Chs6p, and the Chs6p paralogues expressed in baculovirus form a complex of approximately 1 MD that binds synthetic liposomes in a reaction requiring acidic phospholipids, Arf1p, and the nonhydrolyzable GTPγS. The complex remains bound to liposomes centrifuged on a sucrose density gradient. Thin section electron microscopy reveals a spiky coat structure on liposomes incubated with the full complex, Arf1p, and GTPγS. We termed the novel coat exomer for its role in exocytosis from the TGN to the cell surface. Unlike other coats (e.g., coat protein complex I, II, and clathrin/adaptor protein complex), the exomer does not form buds or vesicles on liposomes.

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Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1

eLife ◽

10.7554/elife.02362 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 64

Author(s):

Xiaofei Jia ◽

Erin Weber ◽

Andrey Tokarev ◽

Mary Lewinski ◽

Maryan Rizk ◽

...

Keyword(s):

Cell Surface ◽

Membrane Trafficking ◽

Protein Complex ◽

Adaptor Protein ◽

Structural Basis ◽

Host Proteins ◽

Viral Protein U ◽

Sorting Motif ◽

Clathrin Adaptor ◽

Hiv 1

BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins.

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