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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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Localization of Pmel17 relative to EEA1 and endocytic tracers. (A and B) Ultrathin cryosections of MNT-1 cells were double labeled with HMB50 (anti-Pmel17; PAG 10) and anti-EEA1 (PAG 15). (A) EEA1 is detected in small vesicles (arrowheads) and on the limiting membrane of a compartment that also contains Pmel17 (star). Note that labeling for Pmel17 and EEA1 is segregated (B) EEA1 (PAG 15) is enriched in electron lucent vesicular compartments that are poorly labeled for Pmel17 (PAG 10). Note the unusual morphology of the endosomal compartments containing the bulk of Pmel17 (CE), which are poorly labeled for EEA1 and display on their cytosolic side an electron dense coat (arrowheads). (C) Cryosection of MNT-1 cells exposed to Tf-FITC for 20 min at 37°C, labeled with anti-FITC antibodies and PAG 15. Tf-FITC is present in small tubulovesicular structures (arrowheads) and in Pmel17-positive (PAG 10) coated endosomes (CE). (Inset in C) MNT-1 cells pulsed with BSAG for 5 min and chased for 10 min. BSAG (5 nm) at this time point is mainly detected in the Pmel17-positive coated endosome (CE). Pmel17 was labeled with HMB50 and PAG 15. Bars, 100 nm.
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Figure 4: Localization of Pmel17 relative to EEA1 and endocytic tracers. (A and B) Ultrathin cryosections of MNT-1 cells were double labeled with HMB50 (anti-Pmel17; PAG 10) and anti-EEA1 (PAG 15). (A) EEA1 is detected in small vesicles (arrowheads) and on the limiting membrane of a compartment that also contains Pmel17 (star). Note that labeling for Pmel17 and EEA1 is segregated (B) EEA1 (PAG 15) is enriched in electron lucent vesicular compartments that are poorly labeled for Pmel17 (PAG 10). Note the unusual morphology of the endosomal compartments containing the bulk of Pmel17 (CE), which are poorly labeled for EEA1 and display on their cytosolic side an electron dense coat (arrowheads). (C) Cryosection of MNT-1 cells exposed to Tf-FITC for 20 min at 37°C, labeled with anti-FITC antibodies and PAG 15. Tf-FITC is present in small tubulovesicular structures (arrowheads) and in Pmel17-positive (PAG 10) coated endosomes (CE). (Inset in C) MNT-1 cells pulsed with BSAG for 5 min and chased for 10 min. BSAG (5 nm) at this time point is mainly detected in the Pmel17-positive coated endosome (CE). Pmel17 was labeled with HMB50 and PAG 15. Bars, 100 nm.

Mentions: Double immunogold labeling of ultrathin cryosections of MNT-1 cells reveals that Pmel17 and the early endosomal marker, EEA1 (Mu et al. 1995), are both present within two types of electron-lucent compartments (Fig. 4A and Fig. B). Small vesicles and tubules that are strongly labeled for EEA1 are weakly labeled for Pmel17 (Fig. 4A and Fig. B). These structures likely represent early sorting endosomes, as they were also labeled with Tf-FITC and fluid phase tracers internalized for 10 and 5 min, respectively (see below). Labeling for Pmel17 is more intense in the larger stage I–like structures (Fig. 4A and Fig. B), which are observed in all pigmented melanoma cell lines and those primary melanocytes analyzed and are distinguished by the presence of a planar cytoplasmic coat on their surface (Fig. 4 B; see below). Among these coated compartments, those with fewer internal membranes are often also labeled for EEA1; however, the labeling for Pmel17 and EEA1 is often segregated (Fig. 4 A). Those coated compartments with more internal vesicles and the characteristic stage II premelanosomes (containing the bulk of Pmel17) show only sparse labeling for EEA1 (Fig. 4 A). Quantitation of the immunogold labeling (see Fig. 7 B, bar graph) emphasizes that progressive enrichment for Pmel17 correlates with depletion of EEA1, and suggests that the coated Pmel17–EEA1-positive compartment is an intermediate between early endosomes and stage II premelanosomes. We will refer to this stage I–like compartment as the coated endosome.


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)

Localization of Pmel17 relative to EEA1 and endocytic tracers. (A and B) Ultrathin cryosections of MNT-1 cells were double labeled with HMB50 (anti-Pmel17; PAG 10) and anti-EEA1 (PAG 15). (A) EEA1 is detected in small vesicles (arrowheads) and on the limiting membrane of a compartment that also contains Pmel17 (star). Note that labeling for Pmel17 and EEA1 is segregated (B) EEA1 (PAG 15) is enriched in electron lucent vesicular compartments that are poorly labeled for Pmel17 (PAG 10). Note the unusual morphology of the endosomal compartments containing the bulk of Pmel17 (CE), which are poorly labeled for EEA1 and display on their cytosolic side an electron dense coat (arrowheads). (C) Cryosection of MNT-1 cells exposed to Tf-FITC for 20 min at 37°C, labeled with anti-FITC antibodies and PAG 15. Tf-FITC is present in small tubulovesicular structures (arrowheads) and in Pmel17-positive (PAG 10) coated endosomes (CE). (Inset in C) MNT-1 cells pulsed with BSAG for 5 min and chased for 10 min. BSAG (5 nm) at this time point is mainly detected in the Pmel17-positive coated endosome (CE). Pmel17 was labeled with HMB50 and PAG 15. Bars, 100 nm.
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Figure 4: Localization of Pmel17 relative to EEA1 and endocytic tracers. (A and B) Ultrathin cryosections of MNT-1 cells were double labeled with HMB50 (anti-Pmel17; PAG 10) and anti-EEA1 (PAG 15). (A) EEA1 is detected in small vesicles (arrowheads) and on the limiting membrane of a compartment that also contains Pmel17 (star). Note that labeling for Pmel17 and EEA1 is segregated (B) EEA1 (PAG 15) is enriched in electron lucent vesicular compartments that are poorly labeled for Pmel17 (PAG 10). Note the unusual morphology of the endosomal compartments containing the bulk of Pmel17 (CE), which are poorly labeled for EEA1 and display on their cytosolic side an electron dense coat (arrowheads). (C) Cryosection of MNT-1 cells exposed to Tf-FITC for 20 min at 37°C, labeled with anti-FITC antibodies and PAG 15. Tf-FITC is present in small tubulovesicular structures (arrowheads) and in Pmel17-positive (PAG 10) coated endosomes (CE). (Inset in C) MNT-1 cells pulsed with BSAG for 5 min and chased for 10 min. BSAG (5 nm) at this time point is mainly detected in the Pmel17-positive coated endosome (CE). Pmel17 was labeled with HMB50 and PAG 15. Bars, 100 nm.
Mentions: Double immunogold labeling of ultrathin cryosections of MNT-1 cells reveals that Pmel17 and the early endosomal marker, EEA1 (Mu et al. 1995), are both present within two types of electron-lucent compartments (Fig. 4A and Fig. B). Small vesicles and tubules that are strongly labeled for EEA1 are weakly labeled for Pmel17 (Fig. 4A and Fig. B). These structures likely represent early sorting endosomes, as they were also labeled with Tf-FITC and fluid phase tracers internalized for 10 and 5 min, respectively (see below). Labeling for Pmel17 is more intense in the larger stage I–like structures (Fig. 4A and Fig. B), which are observed in all pigmented melanoma cell lines and those primary melanocytes analyzed and are distinguished by the presence of a planar cytoplasmic coat on their surface (Fig. 4 B; see below). Among these coated compartments, those with fewer internal membranes are often also labeled for EEA1; however, the labeling for Pmel17 and EEA1 is often segregated (Fig. 4 A). Those coated compartments with more internal vesicles and the characteristic stage II premelanosomes (containing the bulk of Pmel17) show only sparse labeling for EEA1 (Fig. 4 A). Quantitation of the immunogold labeling (see Fig. 7 B, bar graph) emphasizes that progressive enrichment for Pmel17 correlates with depletion of EEA1, and suggests that the coated Pmel17–EEA1-positive compartment is an intermediate between early endosomes and stage II premelanosomes. We will refer to this stage I–like compartment as the coated endosome.

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