Sec16A is known to be required for COPII vesicle formation from the ER. Here, Bruno et al. show that, independent of its role at the ER, Sec16A is a RAB10 effector involved in the insulin-stimulated formation of specialized transport vesicles that ferry the GLUT4 glucose transporter to the plasma membrane of adipocytes.

Macrophages use an extracellular, hydrolytic compartment formed by local actin polymerization to digest aggregated LDL (agLDL). Catabolism of agLDL promotes foam cell formation and creates an environment rich in LDL catabolites, including cholesterol and ceramide. Increased ceramide levels are present in lesional LDL, but the effect of ceramide on macrophage proatherogenic processes remains unknown. Here, we show that macrophages accumulate ceramide in atherosclerotic lesions. Using macrophages from sphingosine kinase 2 KO (SK2KO) mice to mimic ceramide-rich conditions of atherosclerotic lesions, we show that SK2KO macrophages display impaired actin polymerization and foam cell formation in response to contact with agLDL. C16-ceramide treatment impaired wild-type but not SK2KO macrophage actin polymerization, confirming that this effect is due to increased ceramide levels. We demonstrate that knockdown of RhoA or inhibition of Rho kinase restores agLDL-induced actin polymerization in SK2KO macrophages. Activation of RhoA in macrophages was sufficient to impair actin polymerization and foam cell formation in response to agLDL. Finally, we establish that during catabolism, macrophages take up ceramide from agLDL, and inhibition of ceramide generation modulates actin polymerization. These findings highlight a critical regulatory pathway by which ceramide impairs actin polymerization through increased RhoA/Rho kinase signaling and regulates foam cell formation.

Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of GLUT4 from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling module is required for insulin-stimulated GLUT4 translocation to the PM, although where it intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to control GLUT4 mobilization from a Trans Golgi Network (TGN) storage compartment, establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN from which newly synthesized lysosomal proteins are targeted to the late endosomes and the ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not mobilize ATP7A nor does copper mobilize GLUT4, and RAB10 is not required for copper-elicited ATP7A mobilization. Consequently, GLUT4 intracellular sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. Our results define the GLUT4-containing region of the TGN as a sorting and storage site from which different cargo are mobilized by distinct signals through unique molecular machinery.

25Insulin controls glucose uptake into muscle and fat cells by inducing a net redistribution of 26 GLUT4 from intracellular storage to the plasma membrane (PM). The TBC1D4-RAB10 signaling 27 module is required for insulin-stimulated GLUT4 translocation to the PM, although where it 28 intersects GLUT4 traffic was unknown. Here we demonstrate that TBC1D4-RAB10 functions to 29 control GLUT4 mobilization from a Trans Golgi Network (TGN) storage compartment, 30 establishing that insulin, in addition to regulating the PM proximal effects of GLUT4-containing 31 vesicles docking to and fusion with the PM, also directly regulates the behavior of GLUT4 32 deeper within the cell. We also show that GLUT4 is retained in an element/domain of the TGN 33 from which newly synthesized lysosomal proteins are targeted to the late endosomes and the 34 ATP7A copper transporter is translocated to the PM by elevated copper. Insulin does not 35 mobilize ATP7A nor does copper mobilize GLUT4. Consequently, GLUT4 intracellular 36 sequestration and mobilization by insulin is achieved, in part, through utilizing a region of the 37 TGN devoted to specialized cargo transport in general rather than being specific for GLUT4. 38Our results define GLUT4-containing region of the TGN as a sorting and storage site from which 39 different cargo are mobilized by distinct signals. 40 11) whose delivery to the PM is regulated by insulin (2). GLUT4 in the PM cycles back to the 51 TGN via the endosomal pathway (2, 12, 13). Targeting GLUT4 from endosomes to the TGN has 52 an important role in basal intracellular GLUT4 retention. Mutations in GLUT4 that disrupt its 53 traffic from endosomes to the TGN are poorly retained in basal conditions and are not properly 54 translocated to the PM upon insulin stimulation (6, 14-16). These results identify the TGN as the 55 site for formation of IRVs. The TGN is a main sorting compartment along the biosynthetic and 56 endocytic pathways. Cargoes to be targeted to distinct destinations are sorted and packaged 57 into the correct transport vesicles in the TGN. The relationship between the TGN containing 58 GLUT4 and the TGN involved in the traffic of other cargoes is not known (2, 6, 8, 12). 59Insulin signaling triggers multiple discrete molecular events that mediate efficient recruitment, 60 docking, and fusion of IRVs with the PM (17-19). These events lead to a decrease in the size of 61 the intracellular GLUT4 pool concomitant with an increase of GLUT4 in the PM. As GLUT4 in 62 the PM is in equilibrium with intracellular GLUT4, endocytosis of GLUT4 dynamically removes 63 GLUT4 from the PM. Thus, maintenance of the insulin-stimulated dynamic increase in GLUT4 64 in the PM requires the continual ferrying of GLUT4-containing IRVs to the PM. Insulin signaling 65 can add to the IRV pool by increasing the rate of GLUT4 mobilization from the TGN in nascent 66IRVs. Despite the biological importance, insulin regulation of GLUT4 trafficking at the 67 perinuclear region has not been thoroughly interrogated. 68A key aspect of insu...

The pathological accumulation of cholesterol is a signature feature of Niemann–Pick type C (NPC) disease, in which excessive lipid levels induce Purkinje cell death in the cerebellum. NPC1 encodes a lysosomal cholesterol-binding protein, and mutations in NPC1 drive cholesterol accumulation in late endosomes and lysosomes (LE/Ls). However, the fundamental role of NPC proteins in LE/L cholesterol transport remains unclear. Here, we demonstrate that NPC1 mutations impair the projection of cholesterol-containing membrane tubules from the surface of LE/Ls. A proteomic survey of purified LE/Ls identified StARD9 as a novel lysosomal kinesin responsible for LE/L tubulation. StARD9 contains an N-terminal kinesin domain, a C-terminal StART domain, and a dileucine signal shared with other lysosome-associated membrane proteins. Depletion of StARD9 disrupts LE/L tubulation, paralyzes bidirectional LE/L motility and induces accumulation of cholesterol in LE/Ls. Finally, a novel StARD9 knock-out mouse recapitulates the progressive loss of Purkinje cells in the cerebellum. Together, these studies identify StARD9 as a microtubule motor protein responsible for LE/L tubulation and provide support for a novel model of LE/L cholesterol transport that becomes impaired in NPC disease.

In this protocol we describe a quantitative biochemical assay to assess the efficiency of endoplasmic reticulum (ER) to Golgi protein transport in adipocytes (Bruno et al., 2016). The assay takes advantage of the fact that adipocytes secrete various bioactive proteins, known as adipokines. As a measure of ER to Golgi flux we determine the rate of bulk secretion of the adipokine adipsin post washout of Brefeldin A (BFA) treatment using immunoblotting. Because BFA treatment results in an accumulation of adipsin in the ER, the exit of adipsin from the ER upon BFA washout is synchronized across cells and experimental conditions. Thus, using this simple assay one can robustly determine if perturbations, such as knocking down a protein, have an effect on ER to Golgi protein transport.