Dynamics of intracellular clathrin/AP1- and clathrin/AP3-containing carriers.

Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits, or dynamin2) with a spatial precision of up to ~30 nm and a temporal resolution of ~1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.

Protein complexes containing CYFIP/Sra/PIR121 coordinate Arf1 and Rac1 signalling during clathrin-AP-1-coated carrier biogenesis at the TGN.

Actin dynamics is a tightly regulated process involved in various cellular events including biogenesis of clathrin-coated, AP-1 (adaptor protein 1)-coated transport carriers connecting the trans-Golgi network (TGN) and the endocytic pathway. However, the mechanisms coordinating coat assembly, membrane and actin remodelling during post-TGN transport remain poorly understood. Here we show that the Arf1 (ADP-ribosylation factor 1) GTPase synchronizes the TGN association of clathrin-AP-1 coats and protein complexes comprising CYFIP (cytoplasmic fragile-X mental retardation interacting protein; Sra, PIR121), a clathrin heavy chain binding protein associated with mental retardation. The Rac1 GTPase and its exchange factor beta-PIX (PAK-interacting exchange factor) activate these complexes, allowing N-WASP-dependent and Arp2/3-dependent actin polymerization towards membranes, thus promoting tubule formation. These phenomena can be recapitulated with synthetic membranes. This protein-network-based mechanism facilitates the sequential coordination of Arf1-dependent membrane priming, through the recruitment of coats and CYFIP-containing complexes, and of Rac1-dependent actin polymerization, and provides complementary but independent levels of regulation during early stages of clathrin-AP1-coated carrier biogenesis.

Protein networks supporting AP-3 function in targeting lysosomal membrane proteins.

The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified approximately 30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles.

The clathrin adaptor Gga2p is a phosphatidylinositol 4-phosphate effector at the Golgi exit.

Phosphatidylinositol 4-phosphate (PI(4)P) is a key regulator of membrane transport required for the formation of transport carriers from the trans-Golgi network (TGN). The molecular mechanisms of PI(4)P signaling in this process are still poorly understood. In a search for PI(4)P effector molecules, we performed a screen for synthetic lethals in a background of reduced PI(4)P and found the gene GGA2. Our analysis uncovered a PI(4)P-dependent recruitment of the clathrin adaptor Gga2p to the TGN during Golgi-to-endosome trafficking. Gga2p recruitment to liposomes is stimulated both by PI(4)P and the small GTPase Arf1p in its active conformation, implicating these two molecules in the recruitment of Gga2p to the TGN, which ultimately controls the formation of clathrin-coated vesicles. PI(4)P binding occurs through a phosphoinositide-binding signature within the N-terminal VHS domain of Gga2p resembling a motif found in other clathrin interacting proteins. These data provide an explanation for the TGN-specific membrane recruitment of Gga2p.

Proteomic analysis of adaptor protein 1A coats selectively assembled on liposomes.

Coat components localize to specific membrane domains, where they sort selected transmembrane proteins. To study how clathrin coats are stabilized on such domains and to identify the protein networks involved, we combined proteomic screens and in vitro liposome-based assays that recapitulate the fidelity of protein sorting in vivo. Our study identifying approximately 40 proteins on AP-1A-coated liposomes revealed that AP-1A coat assembly triggers the concomitant recruitment of Rac1, its effectors, and the Wave/Scar complex as well as that of Rab11 and Rab14. The coordinated recruitment of these different machineries requires a mosaic of membrane components comprising the GTPase ADP-ribosylation factor 1, sorting signals in selected transmembrane proteins, and phosphatidylinositol 4-phosphate. These results demonstrate that the combinatorial use of low-affinity binding sites present on the same membrane domain accounts not only for a selective coat assembly but also for the coordinated assembly of selected machineries required for actin polymerization and subsequent membrane fusion.

Anchorage of synthetic peptides onto liposomes via hydrazone and alpha-oxo hydrazone bonds. preliminary functional investigations.

Synthetic peptidoliposomes have been designed and prepared according to a chemoselective ligation. Two aldehyde-functionalized lipidic anchors were synthesized and incorporated into the lipidic bilayers of unilamellar liposomes during their preparation. Complementary hydrazino acetyl peptides were synthesized on the solid phase using N,N',N'-tri(tert-butyloxycarbonyl)-hydrazino acetic acid and further coupled to the aldehyde groups displayed at the surface of the vesicles. Coupling yields were measured by amino acid hydrolysis following total acid hydrolysis. The ligation methodology proved superior to the simple insertion of lipopeptides, which was performed for comparison in terms of yields, implementation, and reproducibility. To check whether the grafted-peptides were accessible and functional, cytoplasmic sequences of LAMP protein (lysosomal associated membrane protein), which is involved in intracellular membrane trafficking, have been selected. Using this model, we demonstrated in vitro the specific interaction of the synthetic LAMP-peptidoliposomes with the cytoplasmic adaptor protein AP-3, a result that contributes to the understanding of protein sorting in cells. Thus, these results clearly indicate the usefulness of such peptidoliposomes, easily prepared by hydrazone chemoselective ligation, as a tool for biological investigation.