Abstract
Tyrosine-based sorting signals in the cytosolic tails of membrane proteins have been found to bind directly to the medium chain subunit (mu) of the adaptor complexes AP-1 and AP-2. For the leucine-based signals, an interaction with AP-1 and AP-2 has been reported, but no specific interacting subunit has been demonstrated. After searching for molecules interacting with the leucine-based sorting signals within the cytosolic tail of the major histocompatibility complex class II-associated invariant chain using a phage display approach, we identified phage clones with homology to a conserved region of the AP-1 and AP-2 mu chains. To investigate the relevance of these findings, we have expressed regions of mouse mu1 and mu2 chains on phage gene product III and investigated the binding to tail sequences from various transmembrane proteins with known endosomal targeting signals. Enzyme-linked immunosorbent binding assays showed that these phages specifically recognized peptides containing functional leucine- and tyrosine-based sorting signals, suggesting that these regions of the mu1 and mu2 chains interact with both types of sorting signals.
Highlights
The cytosolic tails of membrane proteins contain the information for targeting to various intracellular destinations
Identification of a fUSE5 Clone Binding to an Invariant Chain Tail Peptide—After screening a random fUSE5 display library against a peptide derived from the 15 N-terminal amino acids of invariant chain (Ii) including an LI sorting signal, we identified phagedisplayed peptides with homology to a region of the medium chain adaptor subunits () [20]
FUSE5 Clones Expressing Chain Regions of 24 Amino Acids as gpIII Fusion Proteins—We wanted to investigate the relevance of the homologies between the phage clones and the chains by expressing a region from mouse 1 in fUSE5 gpIII and testing its ability to recognize the invariant chain LI signal
Summary
Phage Library and Bacteria—The 10-mer fUSE5 library with a complexity of 2 ϫ 107 primary phage clones has been described previously [18]. Sletten (Biotechnology Center), showing that similar amounts of peptide were coupled per BSA for the wild type and its corresponding mutant This was done to ensure that lack of/reduced binding in the assays not was due to different amounts of peptides. Banting), and the coating efficiency for the wild type compared with the mutant was measured using horseradish peroxidase-streptavidin These experiments showed that similar binding curves were obtained with free and BSA-coupled peptides and that the two peptides coated the MaxiSorp wells. Several fUSE5 clones displaying peptides with homology to a region of the medium chain adaptor subunit () were isolated after screening for phage recognizing the LI sorting signal of the major histocompatibility complex class II-associated invariant chain These were aligned with the corresponding region of various chains. Binding of biotin-labeled phage was detected using horseradish peroxidase-streptavidin and horseradish peroxidase substrate
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