Abstract. Brefeldin A (BFA) has been reported to block protein transport from the ER and cause disassembly of the Golgi complex. We have examined the effects of BFA on the transport and processing of the vesicular stomatitis virus G protein, a model integral membrane protein. Delivery of G protein to the cell surface was reversibly blocked by 6 #g/ml BFA. Pulse-label experiments revealed that in the presence of BFA, G protein became completely resistant to endoglycosidase H digestion. Addition of sialic acid, a trans-Golgi event, was not observed. Despite processing by cis-and medial Golgi enzymes, G protein was localized by indirect immunofluorescence to a reticular distribution characteristic of the ER. By preventing transport of G protein from the ER with the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or by use of the temperature-sensitive mutant ts045, which is restricted to the ER at 40°C, we showed that processing of G protein occurred in the ER and was not due to retention of newly synthesized Golgi enzymes. Rather, redistribution of preexisting cis and medial Golgi enzymes to the ER occurred as soon as 2.5 min after addition of BFA, and was complete by 10-15 min. Delivery of Golgi enzymes to the ER was energy dependent and occurred only at temperatures >~20°C. BFA also induced retrograde transport of G protein from the medial Golgi to the ER. Golgi enzymes were completely recovered from the ER 10 min after removal of BFA. These findings demonstrate that BFA induces retrograde transport of both resident and itinerant Golgi proteins to the ER in a fully reversible manner.

SUMMARY Human influenza A virus (IAV) vaccination is limited by “antigenic drift,” rapid antibody-driven escape reflecting amino acid substitutions in the globular domain of hemagglutinin (HA), the viral attachment protein. To better understand drift, we used anti-hemagglutinin monoclonal Abs (mAbs) to sequentially select IAV escape mutants. Twelve selection steps, each resulting in a single amino acid substitution in the hemagglutinin globular domain, were required to eliminate antigenicity defined by monoclonal or polyclonal Abs. Sequential mutants grow robustly, showing the structural plasticity of HA, although several hemagglutinin substitutions required an epistatic substitution in the neuraminidase glycoprotein to maximize growth. Selecting escape mutants from parental versus sequential variants with the same mAb revealed distinct escape repertoires, attributed to contextual changes in antigenicity and the mutation landscape. Since each hemagglutinin mutation potentially sculpts future mutation space, drift can follow many stochastic paths, undermining its unpredictability and underscoring the need for drift-insensitive vaccines.

The transporter associated with antigen processing (TAP) transports short peptides from the cytosol to the endoplasmic reticulum, where peptides assemble with class I molecules of the major histocompatibility complex. TAP is comprised of two subunits, termed TAP1 and TAP2. We produced recombinant vaccinia viruses that direct synthesis of the TAP subunits, either individually or together. Virus-encoded TAP is rapidly and efficiently assembled (t1 ⁄2 of 5 min or less) by cells and does not spontaneously assemble in detergent extracts. By confocal immunofluorescence microscopy, TAP1 when expressed alone or with TAP2 is largely, if not exclusively, localized to the endoplasmic reticulum. Metabolic labeling with [2-3 H]mannose demonstrates that TAP1 (but not TAP2) possesses Asn-linked oligosaccharides, but the lack of binding of [ CD8ϩ T cells (T CD8ϩ ) recognize peptides, usually 8 -10 residues in length, bound to major histocompatibility complex (MHC) 1 class I molecules (1). Peptides are predominantly generated from a cytosolic pool of proteins (2, 3). Class I molecules consist of a polymorphic integral membrane glycoprotein (␣ chain) complexed to ␤ 2 -microglobulin, a soluble nonglycosylated protein. Both chains possess NH 2 -terminal hydrophobic sequences that target them co-translationally to the endoplasmic reticulum (ER). Most antigenic peptides, having no such ER insertion sequence, remain sequestered on the cytosolic side of the ER membrane and require a specific transporter, termed TAP (acronymic for transporter associated with antigen processing) to access class I molecules. TAP is produced by the association of two MHC-encoded subunits, termed TAP1 and TAP2 (4 -7). The central importance of TAP in T CD8ϩ responses is most stunningly shown by the severe depletion of T cells in mice with a targeted disruption of the TAP1 gene (8).The TAP genes are members of a large family of integral membrane transporters referred to as ATP binding cassette (ABC) proteins since each has a characteristic sequence associated with ATP binding. ATP hydrolysis is believed to drive the transport of the wide variety of substrates handled by the various family members (9). Typically, ABC transporters are comprised of a single subunit containing two cytosolic ATP binding domains of approximately 300 residues and 12 hydrophobic domains believed to traverse the membrane, with short peptides connecting the hydrophobic domains. The structure of TAP is similar to other ABC proteins, with the most notable difference being its division into two polypeptides, TAP1 and TAP2, each containing a single ATP binding domain and 6 potential transmembrane domains.The past 2 years have witnessed rapid progress in understanding TAP-mediated translocation of peptides. Using semiintact and cell-free systems, the basic requirements for peptide length and sequence have been defined (10 -23). There are still sizable gaps, however, in our knowledge of numerous aspects of TAP, including its assembly, intracellular trafficking, and precise mechanism of fun...