RESUMEN
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.
Asunto(s)
Presentación de Antígeno/inmunología , Antiportadores/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulinas/inmunología , Virus/inmunología , Animales , Retículo Endoplásmico/inmunología , Humanos , Proteínas de Transporte de Membrana , Péptidos/inmunología , Proteínas Virales/inmunologíaRESUMEN
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.
Asunto(s)
Presentación de Antígeno , Antiportadores/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoglobulinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos/genética , Antígenos/metabolismo , Línea Celular , Dimerización , Drosophila melanogaster , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Genes MHC Clase I , Proteínas de Transporte de Membrana , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Ovalbúmina/genética , Ovalbúmina/inmunología , Conformación Proteica , TransfecciónRESUMEN
In human cells the association of MHC class I molecules with TAP is thought to be mediated by a third protein termed tapasin. We now show that tapasin is present in murine TAP-class I complexes as well. Furthermore, we demonstrate that a mutant H-2Dd molecule that does not interact with TAP due to a Glu to Lys mutation at residue 222 of the H chain (Dd(E222K)) also fails to bind to tapasin. This finding supports the view that tapasin bridges the association between class I and TAP and implicates residue 222 as a site of contact with tapasin. The inability of Dd(E222K) to interact with tapasin and TAP results in impaired peptide loading within the endoplasmic reticulum. However, significant acquisition of peptides can still be detected as assessed by the decay kinetics of cell surface Dd(E222K) molecules and by the finding that prolonged viral infection accumulates sufficient target structures to stimulate T cells at 50% the level observed with wild-type Dd. Thus, although interaction with tapasin and TAP enhances peptide loading, it is not essential. Finally, a cohort of Dd(E222K) molecules decays more rapidly on the cell surface compared with wild-type Dd molecules but much more slowly than peptide-deficient molecules. This suggests that some of the peptides obtained in the absence of an interaction with tapasin and TAP are suboptimal, suggesting a peptide-editing function for tapasin/TAP in addition to their role in enhancing peptide loading.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antiportadores/metabolismo , Antígenos H-2/metabolismo , Inmunoglobulinas , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Sitios de Unión/genética , Transporte Biológico Activo , Línea Celular , Membrana Celular/inmunología , Retículo Endoplásmico/inmunología , Retículo Endoplásmico/metabolismo , Antígenos H-2/genética , Antígeno de Histocompatibilidad H-2D , Humanos , Cinética , Sustancias Macromoleculares , Proteínas de Transporte de Membrana , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K. Ohnishi et al., J. Bacteriol. 179:6092-6099, 1997). However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J. Malakooti et al., J. Bacteriol. 176:189-197, 1994). This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA. Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted. Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact. Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S. typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start. When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG. A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start. Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected. The translated intervening sequence is rather divergent between S. typhimurium and E. coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment. The significance of the data for FliO is discussed and compared to the equivalent data for CheA. Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.
Asunto(s)
Proteínas Bacterianas/genética , Codón Iniciador/genética , Proteínas de la Membrana , Biosíntesis de Proteínas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón Iniciador/química , Escherichia coli/genética , Genes Bacterianos/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Proteínas Recombinantes de Fusión , Especificidad de la EspecieRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Salmonella typhimurium/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Prueba de Complementación Genética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Operón , Biosíntesis de Proteínas , Señales de Clasificación de Proteína/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Salmonella typhimurium/genética , Alineación de SecuenciaRESUMEN
The FlgH protein of Salmonella typhimurium, from which the outer membrane L ring of the flagellar basal body is constructed, has a consensus motif (LTG C) 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 [3H]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 [3H]palmitate demonstrated that FlgH incorporated into flagellar structure was also labeled. An in-frame fusion between the leader sequence of the periplasmic protein PeIB 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-copy-number 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 [3H]palmitate at low levels, perhaps through acylation of the N-terminal alpha-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.