Biomedical subjects

Margaret S Robinson

Publications and source records attributed to Margaret S Robinson.

13 recordsLinked to original sources

PubMed2006

Comparative proteomics of clathrin-coated vesicles.

Clathrin-coated vesicles (CCVs) facilitate the transport of cargo between the trans-Golgi network, endosomes, and the plasma membrane. This study presents the first comparative proteomics investigation of CCVs. A CCV-enriched fraction was isolated from HeLa cells and a "mock CCV" fraction from clathrin-depleted cells. We used a combination of 2D difference gel electrophoresis and isobaric tags for relative and absolute quantification (iTRAQ) in conjunction with mass spectrometry to analyze and compare the two fractions. In total, 63 bona fide CCV proteins were identified, including 28 proteins whose association with CCVs had not previously been established. These include numerous post-Golgi SNAREs; subunits of the AP-3, retromer, and BLOC-1 complexes; lysosomal enzymes; CHC22; and five novel proteins of unknown function. The strategy outlined in this paper should be widely applicable as a means of distinguishing genuine organelle components from contaminants.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Functional analysis of AP-2 alpha and mu2 subunits.

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA-resistant alpha and mu2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of alpha, the phosphorylation site of mu2, or the YXXPhi binding site of mu2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of alpha, or mutating the PtdIns(4,5)P2 binding site of mu2, has no apparent effect. However, adding a C-terminal GFP tag to alpha renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo.

Adaptor Protein Complex alpha Subunits↗

PubMed2005

Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

Adaptor Protein Complex 1↗

PubMed2005

The aftiphilin/p200/gamma-synergin complex.

Aftiphilin is a protein that was recently identified in database searches for proteins with motifs that interact with AP-1 and clathrin, but its function is unknown. Here we demonstrate that aftiphilin has a second, atypical clathrin binding site, YQW, that colocalizes with AP-1 by immunofluorescence, and that is enriched in clathrin-coated vesicles (CCVs), confirming that it is a bona fide component of the CCV machinery. By gel filtration, aftiphilin coelutes with two other AP-1 binding partners, p200a and gamma-synergin. Antibodies against any one of these three proteins immunoprecipitate the other two, and knocking down any of the three proteins by siRNA causes a reduction in the levels of the other two, indicating that they form a stable complex. Like AP-1-depleted cells, aftiphilin-depleted cells missort a CD8-furin chimera and the lysosomal enzyme cathepsin D. However, whereas AP-1-depleted cells recycle endocytosed transferrin more slowly than untreated cells, aftiphilin-depleted cells accumulate endocytosed transferrin in a peripheral compartment and recycle it more rapidly. These observations show that in general, the aftiphilin/p200/gamma-synergin complex facilitates AP-1 function, but the complex may have additional functions as well, because of the opposing effects of the two knockdowns on transferrin recycling.

Adaptor Protein Complex 1↗

PubMed2005

Sorting of major cargo glycoproteins into clathrin-coated vesicles.

The AP-1 and AP-2 complexes are the most abundant adaptors in clathrin-coated vesicles (CCVs), but clathrin-mediated trafficking can still occur in the absence of any detectable AP-1 or AP-2. To find out whether adaptor abundance reflects cargo abundance, we used lectin pulldowns to identify the major membrane glycoproteins in CCVs from human placenta and rat liver. Both preparations contained three prominent high molecular-weight proteins: the cation-independent mannose 6-phosphate receptor (CIMPR), carboxypeptidase D (CPD) and low-density lipoprotein receptor-related protein 1 (LRP1). To investigate how these proteins are sorted, we constructed and stably transfected CD8 chimeras into HeLa cells. CD8-CIMPR localized mainly to early/tubular endosomes, CD8-CPD to the trans Golgi network and CD8-LRP1 to late/multivesicular endosomes. All three constructs redistributed to the plasma membrane when clathrin was depleted by siRNA. CD8-CIMPR was also strongly affected by AP-2 depletion. CD8-CPD was moderately affected by AP-2 depletion but strongly affected by depleting AP-1 and AP-2 together. CD8-LRP1 was only slightly affected by AP-2 depletion; however, mutating an NPXY motif in the LRP1 tail caused it to become AP-2 dependent. These results indicate that all three proteins have AP-dependent sorting signals, which may help to explain the relative abundance of AP complexes in CCVs. However, the relatively low abundance of cargo proteins in CCV preparations suggests either that some of the APs may be empty or that the preparations may be dominated by empty coats.

Amino Acid Sequence↗

PubMed2004

EpsinR is an adaptor for the SNARE protein Vti1b.

EpsinR is a clathrin-coated vesicle (CCV)-associated protein that binds to vti1b, suggesting that it may be a vti1b-selective adaptor. Depletion of epsinR to undetectable levels in HeLa cells using siRNA causes vti1b to redistribute from the perinuclear region to the cell periphery, but vti1a also redistributes in epsinR-depleted cells, and both vti isoforms redistribute in AP-1-depleted cells. As a more direct assay for epsinR function, we isolated CCVs from control and siRNA-treated cells and then looked for differences in cargo content. In clathrin-depleted cells, both coat and cargo proteins are greatly reduced in this preparation. Knocking down epsinR causes a approximately 50% reduction in the amount of AP-1 copurifying with CCVs and vice versa, indicating that the two proteins are dependent on each other for maximum incorporation into the coat. In addition, vti1b, but not vti1a, is reduced by >70% in CCVs from both epsinR- and AP-1-depleted cells. Because AP-1 knockdown reduces the amount of epsinR in CCVs, it is possible that its effect on vti1b may be indirect. These findings provide in vivo evidence that epsinR is an adaptor for vti1b, and they also show that CCV isolation can be used as an assay for adaptor function.

Adaptor Proteins, Vesicular Transport↗

PubMed2004

Adaptable adaptors for coated vesicles.

Adaptors select cargo for inclusion into coated vesicles in the late secretory and endocytic pathways. Although originally there were thought to be just two adaptors, AP-1 and AP-2, it is now clear that there are many more: two additional adaptor complexes, AP-3 and AP-4, which might function independently of clathrin; a family of monomeric adaptors, the GGAs; and an ever-growing number of cargo-specific adaptors. The adaptors are targeted to the appropriate membrane at least in part by interacting with phosphoinositides, and, once on the membrane, they form interconnected networks to get different types of cargo into the same vesicle. Adaptors participate in trafficking pathways shared by all cells, and they are also used to generate specialized organelles and to influence cell fate during development.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Clathrin-mediated endocytosis in AP-2-depleted cells.

We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

Adaptor Protein Complex 2↗

PubMed2003

Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin.

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.

ADP-Ribosylation Factors↗

PubMed2003

Genetic and phenotypic analysis of the mouse mutant mh2J, an Ap3d allele caused by IAP element insertion.

Mocha (mh), a mouse model for Hermansky-Pudlak syndrome (HPS), is characterized by platelet storage pool deficiency, pigment dilution, and deafness as well as neurological abnormalities. The trans-Golgi/endosome adaptor-related complex AP-3 is missing in mh mice owing to a deletion in the gene encoding the delta subunit. Mice mutant for a second allele, mh(2J), are as hyperactive as mh, and display both spike wave absence and generalized tonic clonic seizures, but have less coat color dilution, no hearing loss, and no hypersynchronized EEG. Here we show that the mh(2J) mutation is due to an IAP element insertion in the Ap3d gene leading to a C-terminally truncated protein. Despite correct assembly of the AP-3 complex and localization to the trans-Golgi network and endosomes, AP-3 function in neurons remains impaired. While mh mice show a severe reduction of vesicular zinc (TIMM staining) owing to mislocalization and degradation of the Zinc transporter ZnT-3, the TIMM and ZnT-3 staining patterns in mh(2J) varies, with normal expression in hippocampal mossy fibers, but abnormal patterns in neocortex. These results indicate that the N-terminal portion of the delta subunit is sufficient for AP-3 complex assembly and subcellular localization to the TGN/endosomes, while subsequent function is regulated in part by cell-specific interactions with the C-terminal portion.

Animals↗

PubMed2003

Structural basis for binding of accessory proteins by the appendage domain of GGAs.

The Golgi-associated, gamma-adaptin-related, ADP-ribosylation-factor binding proteins (GGAs) and adaptor protein (AP)-1 are adaptors involved in clathrin-mediated transport between the trans-Golgi network and endosomal system. The appendage domains of GGAs and the AP-1 gamma-adaptin subunit are structurally homologous and have been proposed to bind to accessory proteins via interaction with short sequences containing phenylalanines and acidic residues. Here we present the structure of the human GGA1 appendage in complex with its cognate binding peptide from the p56 accessory protein (DDDDFGGFEAAETFD) as determined by X-ray crystallography. The interaction is governed predominantly by packing of the first two phenylalanine residues of the peptide with conserved basic and hydrophobic residues from GGA1. Additionally, several main chain hydrogen bonds cause the peptide to form an additional beta-strand on the edge of the preexisting beta-sheet of the protein. Isothermal titration calorimetry was used to assess the affinities of different peptides for the GGA and gamma-appendage domains.

ADP-Ribosylation Factors↗

PubMed2003

EpsinR: an ENTH domain-containing protein that interacts with AP-1.

We have used GST pulldowns from A431 cell cytosol to identify three new binding partners for the gamma-adaptin appendage: Snx9, ARF GAP1, and a novel ENTH domain-containing protein, epsinR. EpsinR is a highly conserved protein that colocalizes with AP-1 and is enriched in purified clathrin-coated vesicles. However, it does not require AP-1 to get onto membranes and remains membrane-associated in AP-1-deficient cells. Moreover, although epsinR binds AP-1 via its COOH-terminal domain, its NH(2)-terminal ENTH domain can be independently recruited onto membranes, both in vivo and in vitro. Brefeldin A causes epsinR to redistribute into the cytosol, and recruitment of the ENTH domain requires GTPgammaS, indicating that membrane association is ARF dependent. In protein-lipid overlay assays, the epsinR ENTH domain binds to PtdIns(4)P, suggesting a possible mechanism for ARF-dependent recruitment onto TGN membranes. When epsinR is depleted from cells by RNAi, cathepsin D is still correctly processed intracellularly to the mature form. This indicates that although epsinR is likely to be an important component of the AP-1 network, it is not necessary for the sorting of lysosomal enzymes.

Adaptor Proteins, Vesicular Transport↗

PubMed2002

Assembly and function of AP-3 complexes in cells expressing mutant subunits.

The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.

Adaptor Proteins, Vesicular Transport↗