A role for acidic residues in di-leucine motif-based targeting to the endocytic pathway.
Recent reports have suggested that major histocompatibility complex class II molecules load peptide through a specialized compartment of the endocytic pathway and are targeted to this pathway by association with invariant chain (Iip31). Therefore we used a site-directed mutagenesis approach to determine whether Iip31 possesses novel protein targeting signals. Our results indicate that two di-leucine-like pairs mediate Iip31 targeting and that an acidic amino acid residue four or five residues N-terminal to each Iip31 di-leucine-like pair is required for endocytic targeting. Results from additional testing with hybrid Iip31 molecules indicate that the acidic residues N-terminal to di-leucine pairs are critical for accumulation of these molecules in large endocytic vesicles and in some cases provide a structure favorable for internalization. The acidic residues N-terminal to di-leucine pairs are important in some sequence contexts in providing a structure favorable for internalization, whereas in other contexts an acidic residue is critical for targeting to, and formation of, large endocytic vesicles. Although our results do not support the idea that Iip31 possesses unique protein targeting motifs, they do suggest that di-leucine motifs may be recognized as part of a larger secondary structure. In addition, our data imply that the targeting motif requirements for internalization may differ from the requirements for further transport in the endocytic pathway.
Pond
L
L
R.W. Johnson Pharmaceutical Research Institute, San Diego, California 92121, USA.
Kuhn
L A
LA
Teyton
L
L
Schutze
M P
MP
Tainer
J A
JA
Jackson
M R
MR
Peterson
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001693:Biological Transport, Active; D017252:CD3 Complex / Q000235:genetics / Q000378:metabolism; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology*; D006367:HeLa Cells; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007930:Leucine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015398:Signal Transduction; D014162:Transfection
D000944:Antigens, Differentiation, B-Lymphocyte; D017252:CD3 Complex; D015704:CD4 Antigens; D000949:Histocompatibility Antigens Class II; C045084:invariant chain; D007930:Leucine