Functional analysis by site-directed mutagenesis of the complex polymorphism in rat transporter associated with antigen processing.
The transporter associated with Ag processing, TAP, is an endoplasmic reticulum resident heterodimeric member of the ATP-binding cassette transporter family. TAP transports short peptides from cytosol to the endoplasmic reticulum lumen for loading into recently synthesized class I MHC molecules. In the rat, two alleles of the TAP2 chain differ in their permissiveness to the transport of peptides with small hydrophobic, polar, or charged amino acids at the C terminus, and this correlates with differences between the peptide sets loaded into certain class I molecules in vivo. We have used segmental exchanges and site-directed mutagenesis to identify the residues in rat TAP2 responsible for differential transport between the two alleles of peptides terminating above all in the positively charged residue, arginine. Of the 25 residues by which the two functional TAP2 alleles differ, we have localized differential transport of peptides with a C-terminal arginine to two adjacent clusters of exchanges in the membrane domain involving a total of five amino acids. Each cluster, transferred by site-directed mutagenesis from the permissive to the restrictive sequence, can independently confer on TAP a partial ability to transport peptides with arginine at the C terminus. The results suggest that the permissive TAP2-A allele evolved in at least two steps, each partially permissive for peptides with charged C termini.
Deverson
E V
EV
Department of Immunology, The Babraham Institute, Cambridge, United Kingdom. tdeverson@bbsrc.ac.uk
Leong
L
L
Seelig
A
A
Coadwell
W J
WJ
Tredgett
E M
EM
Butcher
G W
GW
Howard
J C
JC
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000502:physiology*; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D002460:Cell Line; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011110:Polymorphism, Genetic; D051381:Rats; D013329:Structure-Activity Relationship; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology
D000071450:ATP Binding Cassette Transporter, Subfamily B, Member 3; D018528:ATP-Binding Cassette Transporters; C489514:Tap2 protein, rat