The Tetraspanin CD63 Regulates ESCRT-Independent and -Dependent Endosomal Sorting during Melanogenesis

Abstract

Summary Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for numerous physiological processes including lysosome-related organelle (LRO) biogenesis. PMEL – a component of melanocyte LROs (melanosomes) – is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.

Silencing of human papillomavirus (HPV) E6/E7 oncogene expression affects both the contents and the amounts of extracellular microvesicles released from HPV‐positive cancer cells

“…46 Rather, exosomal cargo sorting is a directed process and involves the selection of specific proteins, possibly by several mechanisms which are, for example, linked to the syndecan-syntenin-ALIX axis 47 or to the activity of tetraspanins. 48,49 Notably, we did not only observe that Survivin is present in exosome-enriched eMVs but also found that the relative Survivin protein concentrations were higher than in unfractionated whole-cell protein extracts. This property of Survivin was unique for the IAPs tested here and indicates that Survivin is enriched in HeLa exosomes.…”

Ongoing activation of sphingosine 1-phosphate receptors mediates maturation of exosomal multivesicular endosomes

“…We have shown that treatment of cells with either SphK inhibitor HACPT or depletion of S1P 1 receptor or SphK2 caused a strong inhibition of CD63 sorting into exosomal ILVs (Figs 4 and 5). It has recently been reported that CD63 directly participates in ESCRT-independent sorting of the Pmel luminal domain into ILVs, and that in the absence of CD63 the Pmel luminal domain is targeted for ESCRT-dependent degradation 29 .…”

“…Likewise, the melanosomal protein Pmel17 in melanoma cells 27 and major histocompatibility complex class II molecules in activated dendritic cells 28 are inserted into ILVs independently of ubiquitination. In addition, the CD63 itself is required for normal Pmel to be sorted into luminal domain in an ESCRT-independent manner 29 . A factor involved in ESCRT-independent ILV biogenesis has recently been proposed, that is, a sphingolipid metabolite ceramide, which have been demonstrated to spontaneously induce formation of internal vesicles in liposomes in vitro 30 .…”

Resolving sorting mechanisms into exosomes

“…Non-tetraspanin membrane proteins may in this way piggyback onto tetraspanin webs for their sorting into exosomes [8]. Interestingly, the tetraspanin CD63, which is highly enriched in exosomes and considered to be important for chaperoning cargo into exosomes [10], can also be recruited by syntenin [11]. Sorting of tetraspanin webs at endosomes into exosomes could thus, similar to syndecans, be driven by the cytoplasmic adaptor syntenin, and the recruitment by syntenin of tetraspanin webs and syndecan clusters are thus integrated processes (Figure 1).…”