ABSTRACT
ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R approximately 35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1-GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1-GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.
Subject(s)
ADP-Ribosylation Factor 1/metabolism , Coat Protein Complex I/metabolism , GTPase-Activating Proteins/metabolism , Membrane Lipids/metabolism , Unilamellar Liposomes/metabolism , ADP-Ribosylation Factor 1/analysis , Amino Acid Motifs , Coat Protein Complex I/analysis , Diffusion , GTPase-Activating Proteins/analysis , Golgi Apparatus/metabolism , Membrane Lipids/analysis , Optical Tweezers , Protein Binding , Unilamellar Liposomes/analysisABSTRACT
In a P{lArB} enhancer detector collection, a line was found that showed upregulated expression within centrally to posteriorly located germarial cysts. It was inserted in the gammaCOP locus on chromosome 3R. GammaCOP is a component of the COPI coatomer involved in membrane traffic. Most of the other known components of the COPI coatomer also showed higher expression in the posterior half of the germarium. Not only meiotic germline cysts but also migrating follicle cells upregulate the COPI subunits. During embryonic and larval development, the COPI subunits are expressed ubiquitously as expected for genes required for cell viability. In addition, they are strongly expressed in the salivary glands and the proventriculus. Whether tissue-specific transcriptional upregulation of COPI subunits is required for the reorganization of membranous compartments that are needed for the developmental processes that confer cyst polarity and follicle maturation will have to be addressed in a genetic study.
