A Cytoplasmic Sequence in Human Tyrosinase Defines a Second Class of Di-leucine-based Sorting Signals for Late Endosomal and Lysosomal Delivery

Abstract

Distinct cytoplasmic sorting signals target integral membrane proteins to late endosomal compartments, but it is not known whether different signals direct targeting by different pathways. The availability of multiple pathways may permit some cell types to divert proteins to specialized compartments, such as the melanosome of pigmented cells. To address this issue, we characterized sorting determinants of tyrosinase, a tissue-specific resident protein of the melanosome. The cytoplasmic domain of tyrosinase was both necessary and sufficient for internalization and steady state localization to late endosomes and lysosomes in HeLa cells. Mutagenesis of two leucine residues within a conventional di-leucine motif ablated late endosomal localization. However, the properties of this di-leucine-based signal were distinguished from that of CD3␥ by overexpression studies; overexpression of the tyrosinase signal, but not the well characterized CD3␥ signal, induced a 4-fold enlargement of late endosomes and lysosomes and interfered with endosomal sorting mediated by both tyrosine-and other di-leucine-based signals. These properties suggest that the tyrosinase and CD3␥ dileucine signals are distinctly recognized and sorted by distinct pathways to late endosomes in non-pigmented cells. We speculate that melanocytic cells utilize the second pathway to divert proteins to the melanosome.

Regulation of tyrosinase trafficking and processing by presenilins: Partial loss of function by familial Alzheimer's disease mutation

“…On the contrary, Tyrp1 may be transported by an AP3-independent but AP1-dependent mechanism (39,40). Although both Tyr and Tyrp1 contain the dileucine motif, which is required for correct routing of the two molecules (41,42), there is no dileucine sequence in DCT͞Tyrp2 C terminus, and a tyrosinebased signal may be used for its trafficking to melanosomes instead (43). Because all three molecules are affected in PS-null melanocytes, PS appears to act on these substrates at a common step independent of adapter proteins.…”

“…The well-established enzymatic activities of Tyr and related proteins make them unlikely cell signaling molecules. Because the C-terminal sequences of these proteins contain melanosomal targeting signals (41)(42)(43), breakage of the CTFs by ␥-secretase is expected to disrupt their melanosomal localization and melanin synthesis activity. Thus, we favor the idea that ␥-secretase functions here to degrade these membrane proteins.…”

Tyrp1 and Oculocutaneous Albinism Type 3

“…A hexapeptide-sorting signal characterized by a di-leucine motif in tyrosinase is sufficient for its targeting to the melanosome. However, dynamic interplay with an additional sorting signal of the tyrosinebased motif of the Y-X-X-Ø (where the Ø is required to be a bulky hydrophobic amino acid), present distal to the di-leucine motif, is also important in the sorting of tyrosinase to the melanosomal/lysosomal compartments (49,50). Both sorting signals are highly conserved between tyrosinase, Tyrp1 and Tyrp2 [review (47)].…”

“…Tyrosinase and LIMP II share the same D(E)ERXP amino acid sequence preceding the di-leucine signal, and the 2 acidic amino acids in this motif appear essential for binding to AP-3. Moreover, mutagenesis of 2 leucine residues within the di-leucine signaling motif of tyrosinase has been shown to prevent late endosomal localization in HeLa cells transfected with the construct (50). In contrast to tyrosinase, the amino acid sequence of Tyrp1 surrounding its di-leucine signal differs.…”

Structure of Human Tyrosinase Related Protein 1 Reveals a Binuclear Zinc Active Site Important for Melanogenesis

“…R93 is near a cluster of four disulfide bonds in the Cys‐rich subdomain (Figure S9), and replacement by a cysteine could interfere with correct disulfide bond formation or even correct folding. Finally, the P513R mutation is in the cytosolic tail domain, which is critical for targeting melanogenic proteins to the melanosomal membrane 18. The P513R replacement may thus interfere with translocation of TYRP1 to the melanosomal membrane.…”