Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.

The flagellar genes fliO, fliP, fliQ, and fliR of Salmonella typhimurium are contiguous within the fliLMNOPQR operon. They are needed for flagellation but do not encode any known structural or regulatory components. They may be involved in flagellar protein export, which proceeds by a type III export pathway. The genes have been cloned and sequenced. The sequences predict proteins with molecular masses of 13,068, 26,755, 9,592, and 28,933 Da, respectively. All four gene products were identified experimentally; consistent with their high hydrophobic residue content, they segregated with the membrane fraction. From N-terminal amino acid sequence analysis, we conclude that fliO starts immediately after fliN rather than at a previously proposed site downstream. FliP existed in two forms, a 25-kDa form and a 23-kDa form. N-terminal amino acid analysis of the 23-kDa form demonstrated that it had undergone cleavage of a signal peptide-a rare process for prokaryotic cytoplasmic membrane proteins. Site-directed mutation at the cleavage site resulted in impaired processing, which reduced, but did not eliminate, complementation of a fliP mutant in swarm plate assays. A cloned fragment encoding the mature form of the protein could also complement the fliP mutant but did so even more poorly. Finally, when the first transmembrane span of MotA (a cytoplasmic membrane protein that does not undergo signal peptide cleavage) was fused to the mature form of FliP, the fusion protein complemented very weakly. Higher levels of synthesis of the mutant proteins greatly improved function. We conclude that, for insertion of FliP into the membrane, cleavage is important kinetically but not absolutely required.In previous work, we have characterized most of flagellar region IIIb of the chromosome of Salmonella typhimurium, from fliE (encoding a basal-body protein) through fliF (basalbody MS ring), fliG (switch), fliH (export), fliI (export and ATPase), fliJ (unknown function), fliK (hook-length control), fliL (unknown function), fliM (switch), and fliN (switch) (4, 7, 10, 12-14, 19, 22, 30). Only fliO, fliP, fliQ, and fliR were left uncharacterized.The fliO, fliP, fliQ, and fliR genes are contiguous within the fliLMNOPQR operon. Their products have not been found in the flagellar structure (11), and there is no evidence that they are regulatory genes. They may therefore play a role in flagellar assembly rather than in flagellar function, perhaps being involved in the export of external components such as the rod proteins, hook protein, and flagellin. The fliP, fliQ, and fliR genes (as well as several other flagellar genes) have homologs in the gene systems for export of virulence factors in a variety of pathogenic bacteria; for example, they correspond to the spa (surface presentation of antigen) genes spaP, spaQ, and spaR in S. typhimurium and to spa24, spa9, and spa29 in Shigella spp. (6). We describe here the characterization of the S. typhimurium fliO, fliP, fliQ, and fliR genes and their products.Of particular interest was the finding...

Peptide fragments from proteins of intracellular pathogens such as viruses are displayed at the cell surface by MHC class I molecules thus enabling surveillance by cytotoxic T cells. Peptides are produced in the cytosol by proteasomal degradation and translocated into the endoplasmic reticulum by the peptide transporter TAP. Empty MHC class I molecules associate with TAP prior to their acquisition of peptides, a process which is assisted and controlled by a series of chaperones. The first part of this review summarizes our current knowledge of this assembly pathway and describes recent observations that tapasin functions as an endoplasmic reticulum retention molecule for empty MHC class I molecules. To defeat the presentation of virus-derived peptides, several DNA viruses have devised strategies to interfere with MHC class I assembly. Although these evasion strategies have evolved independently and differ mechanistically they often target the same step in this pathway. We compare escape mechanisms of different viruses with particular emphasis on the retention of newly synthesized MHC class I molecules in the endoplasmic reticulum and the inhibition of peptide transport by viral proteins.

The lipopolysaccharide (LPS) O-antigen side chains of Klebsiella serotype O1 have been studied by using mutants selected by resistance to a Klebsiella bacteriophage designated O1-A. Two classes of LPS mutants were identified. The major group (90%) synthesized rough LPS. The remaining 10% of the mutants produced a novel LPS profile that lacked the highest-molecular-weight O-substituted molecules (HMW-LPS) but still produced lower-molecular-weight O-substituted species (LMW-LPS). By using antisera raised against mutant Klebsiella strains and antiserum specific for Pasteurella haemolytica serotype 4, it was demonstrated that HMW-LPS and LMW-LPS contain shared epitopes. HMW-LPS also contained an epitope absent in LMW-LPS. This unique epitope was recognized by a monoclonal antibody (O1-52.6) and appears to be responsible for the serological cross-reaction between the O antigens of Klebsiella O1 and Escherichia coli O19. This HMW-LPS epitope was present in eight other Klebsiella O1 isolates which were examined. Electron microscopy demonstrated that HMW-LPS excluded overlying capsular polysaccharide for a distance of 25 to 40 nm. The distance was reduced to 10 to 18 nm in strains which synthesized only LMW-LPS and to zero in rough LPS strains. The HMW-LPS of Klebsiella O1 was shown to be an important virulence determinant, since this molecule was responsible for the resistance of the bacterium to nonspecific, complement-mediated serum killing.

The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG1C) for lipoylation and signal peptide cleavage. We have confirmed the previous finding (M. Homma, K. Ohnishi, T. Iino, and R. M. Macnab, J. Bacteriol. 169:3617-3624, 1987) that it is synthesized in precursor form and processed to a mature form with an apparent molecular mass of ca. 25 kDa. flgH alleles with an in-frame deletion or a 3 truncation still permitted processing. The deletion permitted partial restoration of motility in complementation tests, whereas the truncation did not. Globomycin, an antibiotic which inhibits signal peptide cleavage of prolipoproteins, caused accumulation of precursor forms of FlgH. When cells transformed with a plasmid containing the flgH gene were grown in the presence of [ 3 H]palmitate, a 25-kDa protein doublet was found to be radiolabeled; its identity as FlgH was confirmed by shifts in mobility when the internally deleted and truncated alleles of the gene were used. Hook-basal body complexes from cells grown in the presence of [3 H]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PelB and the mature FlgH sequence, with the putative N-terminal cysteine replaced by glycine, resulted in production of a fusion protein that was processed to its mature form. With a low-copynumber plasmid, the ability of this pelB-flgH fusion to complement a flgH mutant was poor, but with a high-copy-number plasmid, it was comparable to that of the wild type. Although lacking the N-terminal cysteine and therefore being incapable of lipoylation via a thioether linkage, the mutant protein still incorporated [ 3 H]palmitate at low levels, perhaps through acylation of the N-terminal ␣-amino group. We conclude that FlgH is a lipoprotein and that under normal physiological conditions the lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body. We suggest that the N terminus of FlgH is responsible for anchoring the basal body in the outer membrane and that the C terminus may be responsible for binding to the P ring to form the L,P-ring complex.Flagella are the organelles responsible for motility in many bacteria, with rotation of the helical flagellar filament creating the thrust that propels the cell (for a review, see reference 23). The flagellum possesses three main substructures. The major external structure is the filament. The hook is an elbow-shaped structure that connects the filament to the basal-body rod. The basal body is a system of ring structures mounted on a central rod and anchored in the cell envelope (Fig. 1). In gram-negative bacteria such as Salmonella typhimurium and Escherichia coli, the basal-body MS ring is associated with the cytoplasmic membrane and is the structure onto which the flagellar motor is mounted, the P ring is associated with the periplasmic space and...