ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat

Separate Pathways for Antigen Presentation by CD1 Molecules

AbstractThe ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules

An ACAP1-containing clathrin coat complex for endocytic recycling

Whether coat proteins play a widespread role in endocytic recycling remains unclear. We find that ACAP1, a GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF) 6, is part of a novel clathrin coat complex that is regulated by ARF6 for endocytic recycling in two key physiological settings, stimulation-dependent recycling of integrin that is critical for cell migration and insulin-stimulated recycling of glucose transporter type 4 (Glut4), which is required for glucose homeostasis. These findings not only advance a basic understanding of an early mechanistic step in endocytic recycling but also shed key mechanistic insights into major physiological events for which this transport plays a critical role

A PH Domain in ACAP1 Possesses Key Features of the BAR Domain in Promoting Membrane Curvature

SummaryThe BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending

The Late Stage of COPI Vesicle Fission Requires Shorter Forms of Phosphatidic Acid and Diacylglycerol

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process

The late stage of COPI vesicle fission requires shorter forms of phosphatidic acid and diacylglycerol

Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. Phosphatidic acid (PA) and diacylglycerol (DAG) have been found previously to be required for the fission stage of COPI vesicle formation. Here, we find that PA with varying lipid geometry can all promote early fission, but only PA with shortened acyl chains promotes late fission. Moreover, diacylglycerol (DAG) acts after PA in late fission, with this role of DAG also requiring shorter acyl chains. Further highlighting the importance of the short-chain lipid geometry for late fission, we find that shorter forms of PA and DAG promote the vesiculation ability of COPI fission factors. These findings advance a general understanding of how lipid geometry contributes to membrane deformation for vesicle fission, and also how proteins and lipids coordinate their actions in driving this process

Reconsidering the Barefoot Doctor Programme

This paper examines the widely acclaimed Barefoot Doctor campaign in China. The Barefoot Doctor Campaign has come to symbolize the success of Chinese health care to the extent that it has become a model for WHO public health strategy. Yet little has been done to understand how or whether it worked on the ground and what difficulties and contradictions emerged in its implementation. Using previously unexplored party archives as well as newly collected oral interviews, this paper moves away from a narrow focus on party politics and policy formulation by examining the reality of health care at the local level and the challenges faced by local authorities and individuals as the campaigns evolved

Testing the efficacy of voluntary urban greenhouse gas emissions inventories

Drawing from an original dataset of urban metropolitan carbon footprints, we explore the correlations between national level climate change commitments and subnational level inventories. We ask: Does voluntary reporting allow a city to perform better than national average? Does ambitiousness in commitment have an impact on performance in footprint reduction? Does having long-term commitments affect performance in footprint reduction? Do binding national level commitments (such as those under the Kyoto Protocol) affect performance at the city level in terms of footprint reduction? To provide answers, we synthesize data from the largest repository of voluntary sub-national commitments and actions towards footprint reduction and greenhouse gas inventories from around the world, the Carbonn platform. More than 500 cities report at least one action, commitment or inventory to this database. We find, using a subset of this database, perhaps counter intuitively that cities with more ambitious commitments do not necessarily have steeper reductions in emissions. Our data also suggest that having long-term self-reported goals does not make the cities perform better in terms of footprint reduction. This appears to be true for both government and community commitments reported. Lastly, and positively, our data did reveal a statistically significant effect for cities belonging to countries that had committed to the Kyoto Protocol, suggesting the necessity of binding national (and supranational) climate targets

Integrins traffic rapidly via circular dorsal ruffles and macropinocytosis during stimulated cell migration

In response to growth factor stimulation, integrins transit through recycling endosomes to reach newly forming focal adhesions at the cell’s leading edge