Limits...
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.

Show MeSH

Related in: MedlinePlus

Distribution of TRP1 in the Golgi and associated vesicles. (A) Ultrathin cryosections of MNT-1 cells were labeled with anti-TRP1 and PAG 5. TRP1 is often enriched in one side of the Golgi apparatus. (B and C) Double labeling of MNT-1 cells for TRP1 (PAG 5) and γ-adaptin (PAG 10). TRP1 positive tubules and vesicles are labeled for γ-adaptin (arrowheads). Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195785&req=5

Figure 3: Distribution of TRP1 in the Golgi and associated vesicles. (A) Ultrathin cryosections of MNT-1 cells were labeled with anti-TRP1 and PAG 5. TRP1 is often enriched in one side of the Golgi apparatus. (B and C) Double labeling of MNT-1 cells for TRP1 (PAG 5) and γ-adaptin (PAG 10). TRP1 positive tubules and vesicles are labeled for γ-adaptin (arrowheads). Bars, 100 nm.

Mentions: We next analyzed the distribution of melanosomal integral membrane proteins in pigmented cells. IFM analyses of MNT-1 (Figure S1, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1), and other pigmented melanoma cells and melanocytes (not shown) demonstrated that TRP1 and Pmel17 localized to vesicular structures that only partially overlapped. TRP1 staining was brightest in the cell periphery, whereas vesicles containing Pmel17 were evenly distributed throughout the cell. To correlate these structures with morphologically defined melanosomal stages, we analyzed immunogold-labeled ultrathin cryosections of MNT-1 cells by IEM. The anti-Pmel17 mAb HMB50 primarily labels the lumenal space of hypopigmented compartments similar to stage II premelanosomes (Fig. 2; see also Figure S2 A, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1). Gold particles detecting HMB50 (Fig. 2, A–C) or another anti-Pmel17 mAb, HMB45 (Fig. 2 D), often line the striations and underlying membrane vesicles in these compartments. Additional electron-lucent vesicular structures are labeled to a lesser extent with these antibodies (Fig. 2 D; see below), as are Golgi stacks and small vesicles (see below). In contrast, the anti-TRP1 mAb TA99 labels predominantly stage III and IV melanosomes (Fig. 2A and Fig. B; see also Figure S2 B, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1), mostly on the limiting membrane but occasionally on internal membranes. Labeling for TRP1 is also observed in Golgi stacks and associated vesicles; in contrast to Pmel17, which is distributed randomly throughout the Golgi apparatus (see below), TRP1 is frequently enriched on the trans side, particularly in tubular structures that are often coated (Fig. 3). Most of these coated structures are labeled with antibodies to γ-adaptin (Fig. 3B and Fig. C). Tyrosinase is poorly labeled in MNT-1 cells but codistributes with TRP1, predominantly in stage IV melanosomes (not shown). Similar labeling patterns for TRP1 and Pmel17 are observed in other melanoma cells and melanocytes, as exemplified in primary melanocytes from a pigmented nevus (Fig. 2 B). These results extend previous observations suggesting that Pmel17 is a matrix protein of premelanosomes (Kobayashi et al. 1994; Lee et al. 1996), whereas TRP1 is a component of mature melanosomes.


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)

Distribution of TRP1 in the Golgi and associated vesicles. (A) Ultrathin cryosections of MNT-1 cells were labeled with anti-TRP1 and PAG 5. TRP1 is often enriched in one side of the Golgi apparatus. (B and C) Double labeling of MNT-1 cells for TRP1 (PAG 5) and γ-adaptin (PAG 10). TRP1 positive tubules and vesicles are labeled for γ-adaptin (arrowheads). Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Distribution of TRP1 in the Golgi and associated vesicles. (A) Ultrathin cryosections of MNT-1 cells were labeled with anti-TRP1 and PAG 5. TRP1 is often enriched in one side of the Golgi apparatus. (B and C) Double labeling of MNT-1 cells for TRP1 (PAG 5) and γ-adaptin (PAG 10). TRP1 positive tubules and vesicles are labeled for γ-adaptin (arrowheads). Bars, 100 nm.
Mentions: We next analyzed the distribution of melanosomal integral membrane proteins in pigmented cells. IFM analyses of MNT-1 (Figure S1, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1), and other pigmented melanoma cells and melanocytes (not shown) demonstrated that TRP1 and Pmel17 localized to vesicular structures that only partially overlapped. TRP1 staining was brightest in the cell periphery, whereas vesicles containing Pmel17 were evenly distributed throughout the cell. To correlate these structures with morphologically defined melanosomal stages, we analyzed immunogold-labeled ultrathin cryosections of MNT-1 cells by IEM. The anti-Pmel17 mAb HMB50 primarily labels the lumenal space of hypopigmented compartments similar to stage II premelanosomes (Fig. 2; see also Figure S2 A, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1). Gold particles detecting HMB50 (Fig. 2, A–C) or another anti-Pmel17 mAb, HMB45 (Fig. 2 D), often line the striations and underlying membrane vesicles in these compartments. Additional electron-lucent vesicular structures are labeled to a lesser extent with these antibodies (Fig. 2 D; see below), as are Golgi stacks and small vesicles (see below). In contrast, the anti-TRP1 mAb TA99 labels predominantly stage III and IV melanosomes (Fig. 2A and Fig. B; see also Figure S2 B, available at http://www.jcb.org/cgi/content/full/152/4/809/DC1), mostly on the limiting membrane but occasionally on internal membranes. Labeling for TRP1 is also observed in Golgi stacks and associated vesicles; in contrast to Pmel17, which is distributed randomly throughout the Golgi apparatus (see below), TRP1 is frequently enriched on the trans side, particularly in tubular structures that are often coated (Fig. 3). Most of these coated structures are labeled with antibodies to γ-adaptin (Fig. 3B and Fig. C). Tyrosinase is poorly labeled in MNT-1 cells but codistributes with TRP1, predominantly in stage IV melanosomes (not shown). Similar labeling patterns for TRP1 and Pmel17 are observed in other melanoma cells and melanocytes, as exemplified in primary melanocytes from a pigmented nevus (Fig. 2 B). These results extend previous observations suggesting that Pmel17 is a matrix protein of premelanosomes (Kobayashi et al. 1994; Lee et al. 1996), whereas TRP1 is a component of mature melanosomes.

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