An alternative route

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The melanosomal protein Pmel17 is sorted into intralumenal vesicles by a novel Lumenal Domain-Dependent Pathway that is independent HRS (hepatocyte-growth-factor-regulated tyrosine-kinase substrate) and ESCRTs (endosomal sorting complexes required for transport).

Marks and colleagues have discovered a novel method for sorting cargo into multivesicular bodies (MVBs). The well-known route for sorting to the intralumenal vesicles (ILVs) of MVBs involves protein ubiquitylation, HRS (hepatocyte-growth-factor-regulated tyrosine-kinase substrate) and the ESCRTs (endosomal sorting complexes required for transport). However, in Developmental Cell, they now describe an alternative route to ILVs for the melanosomal protein Pmel17.

During the biogenesis of melanosomes — the lysosome-related organelles of melanocytes in which melanin pigments are synthesized and stored — Pmel17 is incorporated into ILVs. At some time, it is cleaved in its lumenal domain to release a fibrillogenic fragment, and the resulting fibrils function as a matrix for melanin deposition as the melanosome matures.

To understand the role of ILVs in fibril formation, the authors dissected the molecular requirements for Pmel17 sorting to ILVs. They first showed that Pmel17 sorting was relatively insensitive to dominant-negative interference with the HRS–ESCRT pathway and to HRS depletion. In addition, using immunoelectron microscopy, they confirmed the localization of Pmel17 to ILVs in cells with disrupted HRS–ESCRT function. They also showed that ILVs still formed and Pmel17 was still sorted to them following the disruption of HRS–ESCRT function to an extent that was sufficient to block the sorting of ubiquitylated proteins to ILVs. Pmel17 therefore seems to be sorted to ILVs in an HRS- and ESCRT-independent manner.

So, what determinants are responsible for the sorting of Pmel17 to ILVs? To answer this question, Marks and co-workers analysed the movements of ectopically expressed wild-type and variant forms of Pmel17 in HeLa cells, which do not express endogenous Pmel17. Despite this, ectopic Pmel17 expression in HeLa cells still results in its accumulation in ILVs and in fibrils, which indicates that this sorting pathway is conserved in non-melanocytic cells.

Using this approach, they showed that Pmel17 ubiquitylation is not required for its sorting to ILVs; mutating putative ubiquitylation sites in the cytoplasmic domain of Pmel17 did not disrupt its sorting to ILVs. They also showed that the cytoplasmic and transmembrane domains of Pmel17 could be replaced by those of an irrelevant protein with no detrimental effect on its sorting to ILVs. However, another construct that contained the Pmel17 cytoplasmic domain and the transmembrane and lumenal domains of the irrelevant protein was not sorted correctly, and the authors showed that the efficient sorting of Pmel17 to ILVs requires the two N-terminal lumenal subdomains of Pmel17. To their knowledge, “...this is the first conclusive evidence for a role of a lumenal domain in directing cargo to ILVs.”

In the final part of their study, Marks and colleagues showed that Pmel17 localization to ILVs is required for Pmel17 cleavage and subsequent fibril formation. This is the first time that localization to ILVs has been shown to provide a specialized environment for protein processing. This work has therefore identified a novel route to the ILVs of MVBs, and it might be that several ILV populations arise from different ESCRT-dependent and -independent pathways.

Rachel Smallridge

References

Theos, A. C. et al. A lumenal domain-dependent pathway for sorting to intralumenal vesicles of multivesicular endosomes involved in organelle morphogenesis. Dev. Cell 10, 343–354 (2006) | Article | PubMed |
Katzmann, D. J. No ESCRT to the melanosome: MVB sorting without ubiquitin. Dev. Cell 10, 278–280 (2006) | Article | PubMed |