Protein targeting by tyrosine- and di-leucine-based signals: Evidence for distinct saturable components

Targeting of transmembrane proteins to lysosomes, endosomal compartments, or the trans-Golgi network is largely dependent upon cytoplasmically exposed sorting signals. Among the most widely used signals are those that conform to the tyrosine-based motif, YXXempty set (where Y is tyrosine, X is any amino acid, and empty set is an amino acid with a bulky hydrophobic group), and to the di-leucine (or LL) motif. Signals conforming to both motifs have been implicated in protein localization to similar post-Golgi compartments. We have exploited the saturability of sorting to ask whether different YXXempty set or LL signals use shared components of the targeting machinery. Chimeric proteins containing various cytoplasmic domains indoor targeting signals were overexpressed in HeLa cells by transient transfection. Endogenous transferrin receptor and lysosomal proteins accumulated at the cell surface upon overexpression of chimeric proteins containing functional YXXempty set targeting signals, regardless of the compartmental destination imparted by the signal. Furthermore, overexpression of these chimeric proteins compromised YXXempty set-mediated endocytosis and lysosomal delivery. These activities were ablated by mutating the signals or by appending sequences that conformed to the YXXempty set motif but lacked targeting activity. Interestingly, overexpression of chimeric proteins containing cytoplasmic LL signals failed to induce surface displacement of endogenous YXXempty set-containing proteins, but did displace other proteins containing LL motifs. Our data demonstrate that: (a) Protein targeting and internalization mediated by either YXXempty set or LL motifs are saturable processes; (b) common saturable components are used in YXXempty set-mediated protein internalization and targeting to different post-Golgi compartments; and (c) YXXempty set-and LL-mediated targeting mechanisms use distinct saturable components.