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Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.

Raposo G, Tenza D, Murphy DM, Berson JF, Marks MS - J. Cell Biol. (2001)

Bottom Line: Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane.Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells.Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

View Article: PubMed Central - PubMed

Affiliation: Curie Institute, Research Section, Paris, 7505 France.

ABSTRACT
Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

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Mature melanosomes in MNT-1 cells are not accessed by endocytic tracers. (A) BSAG (5-nm) was internalized for 30 min. BSAG is present in coated endosomes and multivesicular bodies (arrowheads) situated near mature melanosomes labeled for TRP1 (PAG 10) but does not label the melanosomes. (B) BSAG (5-nm) internalized for 2 h accumulates in electron-dense lysosomes but not in TRP1-positive (PAG 10) melanosomes (IV). Bars, 100 nm.
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Figure 9: Mature melanosomes in MNT-1 cells are not accessed by endocytic tracers. (A) BSAG (5-nm) was internalized for 30 min. BSAG is present in coated endosomes and multivesicular bodies (arrowheads) situated near mature melanosomes labeled for TRP1 (PAG 10) but does not label the melanosomes. (B) BSAG (5-nm) internalized for 2 h accumulates in electron-dense lysosomes but not in TRP1-positive (PAG 10) melanosomes (IV). Bars, 100 nm.

Mentions: Although the data indicate that melanosomes are not terminal lysosomes, they do not rule out that melanosomes are specialized late endosomes. To define the relationship between melanosomes and the late endocytic pathway, we assessed the accessibility of melanosomes to internalized BSAG at late time points. Similar to the early time points, BSAG could not be detected in melanosomes of any stage even after 25 min or ∼2 h of chase (Fig. 9A and Fig. B), which are the known kinetics of internalization of fluid phase tracers into late endosomes and lysosomes in most cells (Gruenberg and Maxfield 1995). At these time points, BSAG is clearly visualized in late endosomes and lysosomes that are often closely apposed to melanosomes (Fig. 9 B). We considered the possibility that melanosomes are stable organelles, and thus that the majority of them do not access the endocytic pathway during the internalization procedure. However, even after 6 h of internalization, when BSAG is primarily detected in lysosomes, no BSAG is detected in mature melanosomes (not shown). These observations suggest that melanosomal organelles have limited access to fluid phase markers, are not primary endocytic organelles, and have diverged from the late endocytic pathway.


Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.

Raposo G, Tenza D, Murphy DM, Berson JF, Marks MS - J. Cell Biol. (2001)

Mature melanosomes in MNT-1 cells are not accessed by endocytic tracers. (A) BSAG (5-nm) was internalized for 30 min. BSAG is present in coated endosomes and multivesicular bodies (arrowheads) situated near mature melanosomes labeled for TRP1 (PAG 10) but does not label the melanosomes. (B) BSAG (5-nm) internalized for 2 h accumulates in electron-dense lysosomes but not in TRP1-positive (PAG 10) melanosomes (IV). Bars, 100 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195785&req=5

Figure 9: Mature melanosomes in MNT-1 cells are not accessed by endocytic tracers. (A) BSAG (5-nm) was internalized for 30 min. BSAG is present in coated endosomes and multivesicular bodies (arrowheads) situated near mature melanosomes labeled for TRP1 (PAG 10) but does not label the melanosomes. (B) BSAG (5-nm) internalized for 2 h accumulates in electron-dense lysosomes but not in TRP1-positive (PAG 10) melanosomes (IV). Bars, 100 nm.
Mentions: Although the data indicate that melanosomes are not terminal lysosomes, they do not rule out that melanosomes are specialized late endosomes. To define the relationship between melanosomes and the late endocytic pathway, we assessed the accessibility of melanosomes to internalized BSAG at late time points. Similar to the early time points, BSAG could not be detected in melanosomes of any stage even after 25 min or ∼2 h of chase (Fig. 9A and Fig. B), which are the known kinetics of internalization of fluid phase tracers into late endosomes and lysosomes in most cells (Gruenberg and Maxfield 1995). At these time points, BSAG is clearly visualized in late endosomes and lysosomes that are often closely apposed to melanosomes (Fig. 9 B). We considered the possibility that melanosomes are stable organelles, and thus that the majority of them do not access the endocytic pathway during the internalization procedure. However, even after 6 h of internalization, when BSAG is primarily detected in lysosomes, no BSAG is detected in mature melanosomes (not shown). These observations suggest that melanosomal organelles have limited access to fluid phase markers, are not primary endocytic organelles, and have diverged from the late endocytic pathway.

Bottom Line: Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane.Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells.Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

View Article: PubMed Central - PubMed

Affiliation: Curie Institute, Research Section, Paris, 7505 France.

ABSTRACT
Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

Show MeSH
Related in: MedlinePlus