A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu. The secretin encoded by filamentous phage f1 was purified. Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer. The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms).

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624 -630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by ␣-helices and lies on a surface consisting of antiparallel ␤-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.Specific protective immunity results when heat shock protein (HSP) 1 -peptide complexes purified from tumor cells are injected into inbred mice bearing the same specific type of tumor (1-6). The HSP-mediated specific immunogenicity is also seen with virus-infected cells (7-9). This paradigm is the basis for a new therapeutic strategy against human cancers (9, 10). The immune response is directed against peptides noncovalently bound to the HSP and not to the HSP (for reviews, see Refs. 11 and 12)). HSPs that form the immunogenic peptide complexes include gp96 (GRP94) (13) and calreticulin (14), which reside in the endoplasmic reticulum (ER). Cytosolic HSP70-(15, 16) and HSP90-(17) peptide complexes are also immunogenic. The ER-resident chaperone gp96 (GRP94) has been the most extensively studied from an immunological standpoint (13, 18 -21). It is an abundant stress protein that displays dual functionality: it directs peptides into the immune response pathway and it assists in protein folding. The role of gp96 in the immune response is not well understood at the molecular level. gp96 binds a variety of peptides in vitro and in vivo with little or no apparent amino acid sequence specificity (13,(22)(23)(24). gp96 also binds ATP but the role of nucleotide in peptide loading/unloading is unclear (22,25). Highly purified HSP90, the cytosolic paralog of gp96, probably binds and uses ATP (26). This is in contrast to chaperones HSP70 and BiP (ER paralog of HSP70), where it is clear that ATP binding is important for the release of peptide substrates (r...

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Filamentous phage f1 encodes protein IV (pIV), a protein essential for phage morphogenesis that localizes to the outer membrane of Escherichia coli, where it is found as a multimer of 10 to 12 subunits. Introduction of internal His or Strep affinity tags at different sites in pIV interfered with its function to a variable extent. A spontaneous second-site suppressor mutation in gene IV allowed several different insertion mutants to function. The identical mutation was also isolated as a suppressor of a multimerization-defective missense mutation. A high-molecular-mass pIV species is the predominant form of pIV present in cells. This species is stable in 4% sodium dodecyl sulfate at temperatures up to 65 degrees C and is largely preserved at 100 degrees C in Laemmli protein sample buffer containing 4% sodium dodecyl sulfate. The suppressor mutation makes the high-molecular-mass form of wild-type pIV extremely resistant to dissociation, and it stabilizes the high-molecular-mass form of several mutant pIV proteins to extents that correlate with their level of function. Mixed multimers of pIV(f1) and pIV(Ike) also remain associated during heating in sodium dodecyl sulfate-containing buffers. Thus, sodium dodecyl sulfate- and heat-resistant high-molecular-mass pIV is derived from pIV multimer and reflects the physiologically relevant form of the protein essential for assembly-export.