Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex.

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with ␦-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and ␦-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to ␦-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with ␦-COP but also with ␣-COP. Strong interaction with ␣-COP requires the presence of comparable amounts of ⑀-COP or ␤-COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.The structural integrity of membrane-bound organelles requires the recycling of lipids and proteins. Between Golgi and the endoplasmic reticulum (ER) 1 this retrograde transport is mediated by COPI-coated vesicles (1). The COPI coat from yeast and mammals consists of seven COP proteins (␣-, ␤Ј-, ␤-, ␥-, ␦-, ⑀-, and -COP ϭ coatomer) and the small GTPase ARF1 (2, 3). Sorting of cargo proteins to COPI vesicles can be achieved by direct binding of cargo molecules to coatomer (4 -8). This binding is often mediated by short sequence motifs displayed by cargo molecules (6, 9). The efficient sorting of cargo into COPI vesicles depends on GTP hydrolysis by ARF1 facilitated by a specific GTPase-activating proteins (10).After the uncoating, vesicles are ready to fuse with their specific target membrane (11). Fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself comprises two steps, tethering and docking (12). Both steps involve different sets of proteins. Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity. So far, seven different tethering complexes required for at least five different transport steps were characterized in yeast (13)(14)(15)(16)(17)(18)(19)(20). In some cases, mammalian counterparts of yeast tethering factors were identified (reviewed in Ref. 21).The subsequent docking stage involves specific sets of membrane-anchored proteins, the so-called SNARE proteins (22,23). In contrast to the tethering factors, all known SNARE proteins are members of either of three protein families: the syntaxins, the synaptobrevins or VAMPs and the SNAP-25 family members. To induce membrane fusion, SNARE proteins from apposed membranes must interact in trans. The SNARE or SNARE-like proteins involved in fusion at ...

Section: Fig 5 ␦-Cop May Bind Indirectly To Dsl1psupporting

confidence: 80%

Dsl1p, an Essential Component of the Golgi-Endoplasmic Reticulum Retrieval System in Yeast, Uses the Same Sequence Motif to Interact with Different Subunits of the COPI Vesicle Coat

Andag

Schmitt

2003

“…24) chains of AP-3, both of which are homologous to ␤1 and ␤2. ␦-COP is a component of the COPI coat (38) and exhibits ϳ20% identity to 1 and 2. It is currently unknown whether ␦-COP interacts with any signals; however, two-hybrid analyses have shown that it interacts with ␤-COP, the chain of COPI related to ␤1 and ␤2 (38).…”

Section: Discussionmentioning

confidence: 99%

“…␦-COP is a component of the COPI coat (38) and exhibits ϳ20% identity to 1 and 2. It is currently unknown whether ␦-COP interacts with any signals; however, two-hybrid analyses have shown that it interacts with ␤-COP, the chain of COPI related to ␤1 and ␤2 (38). While still fragmentary, all of this evidence suggests that the and ␤ chain 2, 4, 8, 25, 63, 125, 250, and 1250 g/ml) and separated by SDS-PAGE.…”

Section: Discussionmentioning

confidence: 99%

Functional Domain Mapping of the Clathrin-associated Adaptor Medium Chains ॖ1 and ॖ2

Aguilar

Ohno

Roche

et al. 1997

The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes involved in the recognition of sorting signals present within the cytosolic domain of integral membrane proteins. The medium chains of these complexes, 1 and 2, have been implicated in two types of interaction: assembly with the ␤1 and ␤2 chains of the corresponding complexes and recognition of tyrosine-based sorting signals. In this study, we report the results of a structure-function analysis of the 1 and 2 chains aimed at identifying regions of the molecules that are responsible for each of the two interactions. Analyses using the yeast two-hybrid system and proteolytic digestion experiments suggest that 1 and 2 have a bipartite structure, with the amino-terminal one-third (residues 1-145 of 1 and 2) being involved in assembly with the ␤ chains and the carboxyl-terminal two-thirds (residues 147-423 of 1 and 164 -435 of 2) binding tyrosine-based sorting signals. These observations support a model in which the amino-terminal one-third of 2 is embedded within the core of the AP-2 complex, while the carboxyl-terminal two-thirds of the protein are exposed to the medium, placing this region in a position to interact with tyrosine-based sorting signals.The clathrin-associated adaptors AP-1 and AP-2 are heterotetrameric complexes that mediate attachment of clathrin to membranes and recruitment of integral membrane proteins for incorporation into clathrin-coated vesicles (reviewed in Refs. 1 and 2). AP-1 is localized to the trans-Golgi network, where it mediates biosynthetic protein transport to the endosomal/lysosomal system, whereas AP-2 is localized to the plasma membrane and mediates rapid internalization of endocytic receptors. AP-1 is composed of four chains termed ␥ (ϳ91 kDa), ␤1 (ϳ101 kDa), 1 (ϳ47 kDa), and 1 (ϳ19 kDa). The four chains of AP-2 are homologous to those of AP-1 and are known as ␣ (ϳ104 -108 kDa), ␤2 (ϳ104 kDa), 2 (ϳ50 kDa), and 2 (ϳ17 kDa), respectively. The homologous chains of each complex are thought to subserve similar structural and functional roles. Two of the "large" chains, ␥ and ␣, mediate specific targeting of the corresponding adaptors to the trans-Golgi network and the plasma membrane, respectively (3, 4). The other large chains, ␤1 and ␤2, have been implicated in clathrin binding (5-8). A role for the ␣ chain in clathrin binding has also been proposed (9). Recent work has demonstrated that 1 and 2 (known as the "medium" chains) bind tyrosine-based sorting signals present within the cytosolic domain of some integral membrane proteins and thus probably mediate the capture of these proteins into clathrin-coated areas of the membranes (10 -14). The function of the 1 and 2 chains (referred to as the "small" chains) is currently unknown. The overall structure of the adaptor complexes and the arrangement of the different chains within them have been studied using various approaches. Analyses by deep-etch electron microscopy have revealed that the AP-2 complex has a brickshaped core of ϳ8 nm ("head") with two small...

“…Indeed, some subunits of the non-clathrin coat, COPI, are structurally related to subunits of AP-1 and AP-2 (23)(24)(25)(26). In addition, recent studies have identified other mammalian proteins that display significant homology to AP-1 and AP-2 subunits and are components of previously unknown coats.…”

mentioning

confidence: 99%

β3A-adaptin, a Subunit of the Adaptor-like Complex AP-3

Dell’Angelica

Ooi

Bonifacino

1997

136

101

Recent studies have described a widely expressed adaptor-like complex, named AP-3, which is likely involved in protein sorting in exocytic/endocytic pathways. The AP-3 complex is composed of four distinct subunits. Here, we report the identification of one of the subunits of this complex, which we call ␤3A-adaptin. The predicted amino acid sequence of ␤3A-adaptin reveals that the protein is closely related to the neuronspecific protein ␤-NAP (61% overall identity) and more distantly related to the ␤1-and ␤2-adaptin subunits of the clathrin-associated adaptor complexes AP-1 and AP-2, respectively. Sequence comparisons also suggest that ␤3A-adaptin has a domain organization similar to ␤-NAP and to ␤1-and ␤2-adaptins. ␤3A-adaptin is expressed in all tissues and cells examined. Co-purification and co-precipitation analyses demonstrate that ␤3A-adaptin corresponds to the ϳ140-kDa subunit of the ubiquitous AP-3 complex, the other subunits being ␦-adaptin, p47A (now called 3A) and 3 (A or B). ␤3A-adaptin is phosphorylated on serine residues in vivo while the other subunits of the complex are not detectably phosphorylated. ␤3A-adaptin is not present in significant amounts in clathrin-coated vesicles. The characteristics of ␤3A-adaptin reported here lend support to the idea that AP-3 is a structural and functional homolog of the clathrin-associated adaptors AP-1 and AP-2.Cytosolic protein coats function as mediators of vesicle budding and selection of cargo molecules at different stages of the secretory and endocytic pathways (reviewed in Refs. 1 and 2). Among the various protein coats that have been described to date, those containing the protein clathrin are the most extensively characterized (reviewed in Refs. 3-5). Clathrin coats are found in association with the cytosolic aspect of the trans-Golgi network and the plasma membrane, and with coated vesicles that originate from these organelles. In addition to clathrin, these coats contain specific protein complexes known as adaptors or APs. One of these adaptors, AP-1, is a component of trans-Golgi network clathrin coats and consists of four subunits: ␥-and ␤1-adaptins (ϳ100 kDa), 1 (ϳ47 kDa), and 1 (ϳ19 kDa). Another adaptor, AP-2, is associated with plasma membrane clathrin coats and is also composed of four subunits: ␣-and ␤2-adaptins (ϳ100 kDa), 2 (ϳ50 kDa), and 2 (ϳ17 kDa). The analogous subunits of AP-1 and AP-2 display significant homology to each other; in addition, the adaptor complexes themselves exhibit a similar overall structure of a "head" with two protruding "ears," each separated from the head by a flexible "hinge" (3-5, 46).A major function of AP-1 and AP-2 is to link clathrin lattices to the corresponding membranes. This role is fulfilled by the ␥-and ␤1-adaptin subunits of AP-1 and the ␣-and ␤2-adaptin subunits of AP-2 (6 -9). The adaptors are also responsible for the recognition of sorting signals present in the cytosolic domains of integral membrane proteins (10 -21), an event that leads to the concentration of these proteins within clathrincoated ...