ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Δ synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 null allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Δ mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro–α-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Δ cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.

Ebola virus (EBOV) remains one of the most lethal transmissible infections and is responsible for high fatality rates and substantial morbidity during sporadic outbreaks. With increasing human incursions into endemic regions and the reported possibility of airborne transmission, EBOV is a high-priority public health threat for which no preventive or therapeutic options are currently available. Recent studies have demonstrated that cocktails of monoclonal antibodies are effective at preventing morbidity and mortality in nonhuman primates (NHPs) when administered as a post-exposure prophylactic within 1 or 2 days of challenge. To test whether one of these cocktails (MB-003) demonstrates efficacy as a therapeutic (after the onset of symptoms), we challenged NHPs with EBOV and initiated treatment upon confirmation of infection according to a diagnostic protocol for U.S. Food and Drug Administration Emergency Use Authorization and observation of a documented fever. Of the treated animals, 43% survived challenge, whereas both the controls and all historical controls with the same challenge stock succumbed to infection. These results represent successful therapy of EBOV infection in NHPs. INTRODUCTIONSince its discovery and initial characterization in the mid-1970s, Ebola virus (EBOV; formerly known as Zaire ebolavirus; genus: Ebolavirus, family: Filoviridae) has remained one of the most virulent and deadly pathogens known. With mortality rates approaching 90%, the virus quickly overwhelms the host, inducing a severe hemorrhagic fever and often death during sporadic outbreaks (1, 2). There are currently no licensed vaccines or therapeutics to prevent or treat infection with EBOV or any filovirus. With the increasing ease and speed of global travel and the potential for viral spread via the aerosol route (3), EBOV is a potential public health threat (4). Classification by the Centers for Disease Control as a category A agent also designates EBOV as a bioterrorism threat, making this virus a biodefense research priority (5).Research has identified phosphorodiamidate morpholino oligomers (PMOs), small interfering RNAs (siRNAs), and a vesicular stomatitis virus (VSV)-based vaccine as potential candidates for post-exposure treatment (6-8). These candidates have shown promising efficacy in reducing mortality when administered to nonhuman primates (NHPs) up to 1 hour after exposure. More recently, antibodies were demonstrated to be highly effective in post-exposure prophylaxis of NHPs against EBOV. Passive transfer of macaque hyperimmune globulin was shown to protect rhesus macaques when dosing began 2 days after exposure (9). Similarly, a cocktail of three murine monoclonal antibodies (mAbs) provided 100 and 50% efficacy in cynomolgus macaques when dosing began 1 or 2 days after exposure, respectively (10). Finally, a cocktail of three mAbs with human constant regions (MB-003) manufactured in Nicotiana benthamiana (11) provided 100 or 67% protection in the rhesus macaque model when treatment began 1 hour or 2 days after expo...

These results indicate that Swa2p is a clathrin-binding protein required for normal clathrin function in vivo. They suggest that Swa2p is the yeast ortholog of auxilin and has a role in disassembling clathrin, not only in uncoating clathrin-coated vesicles but perhaps in preventing unproductive clathrin assembly in vivo.

The initial assembly of apolipoprotein B100 (apoB) into lipoprotein particles occurs cotranslationally. To examine steps required to initiate this process, the intracellular folding and assembly of the amino-terminal 28% of apoB (apoB28) was examined using several criteria including nonreducing gel electrophoresis, sensitivity to dithiothreitol (DTT)-mediated reduction, and buoyant density gradient centrifugation. In hepatoma cells, after a 1-min pulse with radiolabeled amino acids, labeled apoB28 migrated during gel electrophoresis in the folded position and was resistant to reduction in vivo with 2 mM DTT. A similar rate and extent of folding was observed in Chinese hamster ovary cells, a microsomal triglyceride transfer protein (MTP)-negative cell line that can neither lipidate nor efficiently secrete apoB28. Amino-terminal folding of apoB28 was essential for its subsequent intracellular lipidation as apoB28 synthesized in hepatoma cells under reducing conditions remained lipid poor (d > 1.25 g/ml) and was retained intracellularly. Upon DTT removal, reduced apoB28 underwent a process of rapid (t1/2 approximately 2 min) post-translational folding followed by a slower process of MTP-dependent lipidation. As with the cotranslational assembly pathway, post-translational lipidation of apoB28 displayed a strict dependence upon amino-terminal folding. We conclude that: 1) folding of the amino-terminal disulfide bonded domain of apoB is achieved prior to the completion of translation and is independent of MTP and events associated with buoyant lipoprotein formation and 2) domain-specific folding of apoBs amino-terminal region is required to initiate MTP-dependent lipid transfer to nascent apoB in the hepatic endoplasmic reticulum.