PROTEIN TARGETING BY TYROSINE-BASED AND DI-LEUCINE-BASED SIGNALS - EVIDENCE FOR DISTINCT SATURABLE COMPONENTS

Targeting of transmembrane proteins to lysosomes, endosomal compartmen ts, or the trans-Golgi network is largely dependent upon cytoplasmical ly exposed sorting signals. Among the most widely used signals are tho se 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 b ulky 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 saturabilit y of sorting to ask whether different YXXempty set or LL signals use s hared components of the targeting machinery. Chimeric proteins contain ing various cytoplasmic domains indoor targeting signals were overexpr essed in HeLa cells by transient transfection. Endogenous transferrin receptor and lysosomal proteins accumulated at the cell surface upon o verexpression of chimeric proteins containing functional YXXempty set targeting signals, regardless of the compartmental destination imparte d by the signal. Furthermore, overexpression of these chimeric protein s compromised YXXempty set-mediated endocytosis and lysosomal delivery . These activities were ablated by mutating the signals or by appendin g sequences that conformed to the YXXempty set motif but lacked target ing activity. Interestingly, overexpression of chimeric proteins conta ining cytoplasmic LL signals failed to induce surface displacement of endogenous YXXempty set-containing proteins, but did displace other pr oteins containing LL motifs. Our data demonstrate that: (a) Protein ta rgeting and internalization mediated by either YXXempty set or LL moti fs are saturable processes; (b) common saturable components are used i n YXXempty set-mediated protein internalization and targeting to diffe rent post-Golgi compartments; and (c) YXXempty set-and LL-mediated tar geting mechanisms use distinct saturable components.