No abstract

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

How the principal functions of the Golgi apparatus - protein processing, lipid synthesis, and sorting of macromolecules - are integrated to constitute cargo-specific trafficking pathways originating from the trans Golgi network (TGN) is unknown. Here we show that the activity of the Golgi localized SPCA1 calcium pump couples sorting and export of secreted proteins to synthesis of new lipid in the TGN membrane. A secreted Ca2+-binding protein, Cab45, constitutes the core component of a Ca2+-dependent, oligomerization-driven sorting mechanism whereby secreted proteins bound to Cab45 are packaged into a TGN-derived vesicular carrier whose membrane is enriched in sphingomyelin, a lipid implicated in TGN-to-cell surface transport. SPCA1 activity is controlled by the sphingomyelin content of the TGN membrane, such that local sphingomyelin synthesis promotes Ca2+ flux into the lumen of the TGN, which drives secretory protein sorting and export, thereby establishing a protein-and lipid-specific secretion pathway.

Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.

One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.sphingomyelin | Golgi apparatus | secretion | equinatoxin A mple evidence indicates that proteins are sorted in the the trans Golgi network (TGN) into distinct types of Golgiderived transport carriers (1), but little is known regarding the lipid content of different carriers. The most abundant sphingolipid, sphingomyelin (SM), is a principal component of the plasma membrane that is synthesized on the luminal membrane leaflets of TGN membranes and transported to the plasma membrane via an uncharacterized pathway. Inhibition of SM synthesis has been reported to slow Golgi-to-plasma membrane trafficking of vesicular stomatitis virus G protein, influenza hemagglutinin, and pancreatic adenocarcinoma up-regulated factor (2-6), suggesting that the SM biosynthetic pathway is broadly required for secretory competence, but the underlying mechanisms are unknown. Furthermore, it remains unclear whether SM trafficking per se, or the activities of SM metabolites such as ceramide and diacylglycerol (DAG), are harnessed for the production of secretory vesicles.Many investigations of intracellular sphingolipid sorting use synthetic short-chain ceramides that are labeled with a fluorescent moiety that can be metabolized, albeit at slow, nonphysiological rates, to short-chain fluorescent SM and glucosylceramide (7-9). In one of the first studies of SM sorting in a polarized epithelial cell line incubated with fluorescent short-chain ceramide, fluorescently labeled lipids accumulated to a higher level in the apical membrane domain compared with the basolateral domain, suggesting that the fluorescently labeled sphingolipids are enriched in apically targeted secretory vesicles (9). A study of secretory vesicle lipid content of yeast (Saccharomyces cerevisiae) cells, which produce mannosylated sphingolipids (but not SM) and ergosterol (but not cholesterol), found that two types of immunopurified secretory vesicles do not differ in terms of abundances of different lipid species (10, 11). Thus, the extent to which lipid sorting occurs in the TGN ...