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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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Quantitative analysis of the distribution of Pmel17 relative to TRP1, EEA1, and LAMP1 in MNT-1 cells. Gold particles labeling either Pmel17 or (A) TRP1, (B) EEA1, or (C) TRP1 or LAMP1 were counted in the different endosomal, melanosomal, or lysosomal compartments in double- (A and B) or triple- (C) labeled ultrathin cryosections of MNT-1 cells. For each set, 30 cell profiles were counted in each of two experiments. Results represent a percentage of the total number of counted gold particles for each marker in the distinct morphologically defined compartments. Only the compartments shown in each experiment were taken into account.
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Figure 7: Quantitative analysis of the distribution of Pmel17 relative to TRP1, EEA1, and LAMP1 in MNT-1 cells. Gold particles labeling either Pmel17 or (A) TRP1, (B) EEA1, or (C) TRP1 or LAMP1 were counted in the different endosomal, melanosomal, or lysosomal compartments in double- (A and B) or triple- (C) labeled ultrathin cryosections of MNT-1 cells. For each set, 30 cell profiles were counted in each of two experiments. Results represent a percentage of the total number of counted gold particles for each marker in the distinct morphologically defined compartments. Only the compartments shown in each experiment were taken into account.

Mentions: Although Pmel17 and TRP1 are highly enriched in stage II and IV melanosomes, respectively, both proteins can be detected in all melanosomal stages. Therefore, we quantitated the immunolabeling for Pmel17 and TRP1 in melanosomes of different stages (see Fig. 7 A, bar graph). Pmel17 is first enriched in stage I–like structures, as described in more detail below, and accumulates to a higher extent in stage II premelanosomes. In contrast, TRP1 is almost undetectable in stage I structures, and only a small percentage of labeling is observed in stage II. Although stage III and particularly stage IV melanosomes are densely labeled for TRP1, the levels of detectable Pmel17 strongly decrease in these structures. These observations stress that premelanosomes and mature melanosomes are enriched in different subsets of resident melanogenic proteins, but that they may represent endpoints of a progression involving gradual loss of some components, such as Pmel17, and acquisition of other components, such as TRP1 and tyrosinase.


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)

Quantitative analysis of the distribution of Pmel17 relative to TRP1, EEA1, and LAMP1 in MNT-1 cells. Gold particles labeling either Pmel17 or (A) TRP1, (B) EEA1, or (C) TRP1 or LAMP1 were counted in the different endosomal, melanosomal, or lysosomal compartments in double- (A and B) or triple- (C) labeled ultrathin cryosections of MNT-1 cells. For each set, 30 cell profiles were counted in each of two experiments. Results represent a percentage of the total number of counted gold particles for each marker in the distinct morphologically defined compartments. Only the compartments shown in each experiment were taken into account.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195785&req=5

Figure 7: Quantitative analysis of the distribution of Pmel17 relative to TRP1, EEA1, and LAMP1 in MNT-1 cells. Gold particles labeling either Pmel17 or (A) TRP1, (B) EEA1, or (C) TRP1 or LAMP1 were counted in the different endosomal, melanosomal, or lysosomal compartments in double- (A and B) or triple- (C) labeled ultrathin cryosections of MNT-1 cells. For each set, 30 cell profiles were counted in each of two experiments. Results represent a percentage of the total number of counted gold particles for each marker in the distinct morphologically defined compartments. Only the compartments shown in each experiment were taken into account.
Mentions: Although Pmel17 and TRP1 are highly enriched in stage II and IV melanosomes, respectively, both proteins can be detected in all melanosomal stages. Therefore, we quantitated the immunolabeling for Pmel17 and TRP1 in melanosomes of different stages (see Fig. 7 A, bar graph). Pmel17 is first enriched in stage I–like structures, as described in more detail below, and accumulates to a higher extent in stage II premelanosomes. In contrast, TRP1 is almost undetectable in stage I structures, and only a small percentage of labeling is observed in stage II. Although stage III and particularly stage IV melanosomes are densely labeled for TRP1, the levels of detectable Pmel17 strongly decrease in these structures. These observations stress that premelanosomes and mature melanosomes are enriched in different subsets of resident melanogenic proteins, but that they may represent endpoints of a progression involving gradual loss of some components, such as Pmel17, and acquisition of other components, such as TRP1 and tyrosinase.

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