Recruitment of coat proteins to liposomes and peptidoliposomes.

Intracellular transport within the cell is generally mediated by membrane vesicles. Their formation is typically initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo and accessory proteins, and deform the lipid bilayer to produce a transport vesicle. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. Here we describe the use of liposomes and peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1 and requires specific lipids and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin. Without cargo peptides, accessory proteins, such as amphiphysin 2, can be identified that stabilize AP-1 binding to liposomal membranes.

Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

Recruitment of coat proteins to peptidoliposomes.

Intracellular transport between compartments within the cell is generally mediated by membrane vesicles. Their formation is initiated by activation of small GTPases that then recruit cytosolic proteins to the membrane surface to form a coat, interact with cargo proteins, and deform the lipid bilayer. Liposomes proved to be a useful tool to study the molecular mechanisms of these processes in vitro. To analyze the involvement of membrane proteins, the cytosolically exposed sequences may be coupled chemically to reactive lipids in the membrane. Here we describe the use of such peptidoliposomes presenting lipid-coupled cytosolic tails of cargo proteins for the in vitro analysis of the membrane recruitment of AP-1 adaptors in the process of forming AP-1/clathrin coats. AP-1 recruitment is mediated by the GTPase Arf1, requires specific lipids, and cargo signals. Interaction with cargo induces AP-1 oligomerization already in the absence of clathrin.

Proprotein convertase PC3 is not a transmembrane protein.

Proprotein convertase PC3 (also known as PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Lacking any extended hydrophobic segments, PC3 was considered to be a secretory protein only peripherally attached to the granule membrane. Recently, evidence has been presented that PC3 is a transmembrane protein with a 115-residue cytoplasmic domain and a membrane-spanning segment containing eight charged amino acids [Arnaoutova, I., et al. (2003) Biochemistry 42, 10445-10455]. Here, we analyzed the membrane topology of PC3 and of a PC3 construct containing a conventional transmembrane segment of 19 leucines. Alkaline extraction was performed to assess membrane integration. Exposure to the cytosol or to the ER lumen was tested by addition of C-terminal tags for phosphorylation or glycosylation, respectively. Protease sensitivity was assayed in permeabilized cells. The results show that the C-terminus of PC3 is translocated across the endoplasmic reticulum membrane. Furthermore, the proposed transmembrane segment of PC3 and a similar one of carboxypeptidase E did not stop polypeptide translocation when inserted into a stop-transfer tester construct. PC3 is thus not a transmembrane protein. These results have implications for the mechanism of granule sorting of PC3 as well as for the topology of PC2 and carboxypeptidase E, which have been reported to span the lipid membrane by homologous charged sequences.