6,849 research outputs found
A role for amphiphysin in AP-1/clathrin coat formation
No full textTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. Clathrin, in combination with different adaptor proteins, is the major coat protein for vesicle formation at the plasma membrane, endosomes, and the trans-Golgi network (TGN). Best characterized is the formation of clathrin coats for endocytosis at the plasma membrane involving the adaptor protein complex AP-2. Clathrin and AP-2 were shown to be at the centre of a complex interactome of proteins accessory to vesicle formation. Considerably less is known about the formation of clathrin coated carriers at the TGN and endosomes, where the adaptor protein complex AP-1 plays a major role.
In vitro studies showed the minimal requirements for association of AP-1 to liposomal membranes to be activated ARF1, phosphoinositides, and either sorting signals or unknown cytosolic factors. We have used a liposome floatation assay to identify cytosolic proteins collaborating with AP-1 at the membrane. Separation of proteins from bovine brain cytosol with several chromatographic methods yielded an active fraction containing amphiphysin 1, amphiphysin 2, and endophilin A1. All three proteins are expressed in brain and known to be involved in AP-2/clathrin coat formation. They consist of an N-terminal N-BAR (Bin, amphiphysin, Rvs) domain for dimerization and membrane binding and a C-terminal SH3 (Src homology 3) domain for interaction with dynamin and synaptojanin. Amphiphysin 1 and 2 in addition contain a middle domain with binding sites for adaptors and clathrin. It was proposed that amphiphysins and endophilin are targeted to membranes with high curvature, such as the neck of a forming vesicle, where they recruit dynamin and synaptojanin in preparation for vesicle fission and uncoating.
In this thesis, I bacterially expressed and purified all three proteins and tested them in the floatation assay for AP-1 membrane binding activity. Only amphiphysin 2 showed activity, both as a homodimer and as a heterodimer with amphiphysin 1. Activity depended on a motif that was shown to bind to AP-1, AP-2, and clathrin in GST pull-down experiments.
Endogenous amphiphysins in primary neurons, as well as transiently expressed in neuronal or fibroblast cell lines, co-localized with AP-1 at the TGN. In addition, when expressed at high levels in neuronal cells, amphiphysins aggregated and interfered dominantly with the TGN localization of AP-1. Both phenomena depended on the presence of the clathrin and adaptor interaction sequence in the amphiphysins. Furthermore, both amphiphysins could be cross-linked to AP-1 in vivo.
Our results indicate that amphiphysin 1 and 2 function not only in clathrin coated vesicle formation for endocytosis at the plasma membrane, but are also part of the machinery forming AP-1/clathrin coats at the TGN and endosomes. This suggests that the machineries for CCV formation with AP-1 and AP-2 at different locations in the cell share more components than previously anticipated
A cytosolic factor mediating membrane recruitment of AP-1 clathrin adaptors
No full textTransport of cargo within the endocytic and secretory pathway is generally mediated by coated vesicles. These vesicles are formed through the recruitment of cytosolic coat proteins to the donor membrane that act as a scaffold to form coated buds and vesicles. At the same time they selectively concentrate cargo proteins by interacting with cytosolic signals. Clathrin, in combination with different adaptor proteins (APs), is the major coat protein for vesicle formation at the plasma membrane, endosomes and the trans-Golgi network. Best characterized is clathrin mediated endocytosis at the plasma membrane which involves AP-2 and a network of associated proteins. Much less is known about AP-1 mediated clathrin coated vesicle formation at the TGN/endosomes. In vitro studies demonstrated that the minimal requirements to recruit AP-1 to liposome membranes are activated Arf1, phosphoinositides, and either sorting signals or an unknown cytosolic factor. In order to identify this factor, we fractionated calf brain cytosol by several chromatographic methods. Fractions were tested for factor dependent AP-1 recruitment activity using an in vitro assay. Purification via ammonium sulfate precipitation, hydrophobic interaction chromatography, anion/cation exchange chromatography or hydroxyapatite chromatography produced a final fraction containing three major proteins: amphiphysin 1, amphiphysinand endophilin A1. All three proteins are known accessory factors in clathrin coated vesicle formation at the plasma membrane. Co-immunodepletion of amphiphysinandresulted in a strong reduction of AP-1 recruitment activity. Therefore we conclude that a heterodimer of amphiphysinandis the long searched for cytosolic factor, required to recruit AP-1 in the absence of sorting signals in vitro. Our results strongly suggest that amphiphysin 1, amphiphysinand endophilin A1 are also involved in AP-1 mediated clathrin coated vesicle formation at the TGN and endosomes in vivo
The Treatment of Ties in AP Correlation
No full textThe Kendall tau and AP correlation coefficients are very commonly use to compare two rankings over the same set of items. Even though Kendall tau was originally defined assuming that there are no ties in the rankings, two alternative versions were soon developed to account for ties in two different scenarios: measure the accuracy of an observer with respect to a true and objective ranking, and measure the agreement between two observers in the absence of a true ranking. These two variants prove useful in cases where ties are possible in either ranking, and may indeed result in very different scores. AP correlation was devised to incorporate a top-heaviness component into Kendall tau, penalizing more heavily if differences occur between items at the top of the rankings, making it a very compelling coefficient in Information Retrieval settings. However, the treatment of ties in AP correlation remains an open problem. In this paper we fill this gap, providing closed analytical formulations of AP correlation under the two scenarios of ties contemplated in Kendall tau. In addition,we developed an R package that implements these coefficients.Best Short Paper Accepted author manuscriptMultimedia ComputingWeb Information System
- …
