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Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.

Leonhardt RM, Vigneron N, Rahner C, Van den Eynde BJ, Cresswell P - J. Biol. Chem. (2010)

Bottom Line: Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment.As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell.Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06519, USA. ralf.leonhardt@yale.edu

ABSTRACT
Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

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The NTR is required for normal subcellular targeting of Pmel17. A, schematic representation of the ΔNTR construct. B, ΔNTR can be processed by pPCs. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS/1% β-mercaptoethanol plus protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C, ΔNTR shows both accelerated pPC processing and turnover. Cells from Fig. 1B, were pulse-labeled for 30 min with 35S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS under vigorous vortexing for 30 min, and analyzed by autoradiography (top panel). A 2-day exposure is shown for the upper part of the gel separated by the dashed line from a 5-day exposure for the lower part of the same gel. Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown (bottom panel). D, newly synthesized ΔNTR localizes to the ER and Golgi. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). E, folded HMB50-reactive ΔNTR co-localizes with Golgi and early endosomal markers. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle markers giantin (ab24586) (Golgi) or EEA1 (ab2900) (early endosomes). F, ΔNTR fails to migrate into LAMP1-positive compartments. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle marker LAMP1 (H4A3) (lysosomes/melanosomes).
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Figure 1: The NTR is required for normal subcellular targeting of Pmel17. A, schematic representation of the ΔNTR construct. B, ΔNTR can be processed by pPCs. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS/1% β-mercaptoethanol plus protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C, ΔNTR shows both accelerated pPC processing and turnover. Cells from Fig. 1B, were pulse-labeled for 30 min with 35S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS under vigorous vortexing for 30 min, and analyzed by autoradiography (top panel). A 2-day exposure is shown for the upper part of the gel separated by the dashed line from a 5-day exposure for the lower part of the same gel. Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown (bottom panel). D, newly synthesized ΔNTR localizes to the ER and Golgi. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). E, folded HMB50-reactive ΔNTR co-localizes with Golgi and early endosomal markers. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle markers giantin (ab24586) (Golgi) or EEA1 (ab2900) (early endosomes). F, ΔNTR fails to migrate into LAMP1-positive compartments. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle marker LAMP1 (H4A3) (lysosomes/melanosomes).

Mentions: The organization of domains within Pmel17 has recently been described, and the first steps have been undertaken to investigate their function in the molecule. In particular, all three large domains within Mα (namely the N-terminal region (NTR, ∼aa 22–210), the polycystic kidney disease-like domain (PKD, ∼aa 211–293), and the repeat domain (RPT, ∼aa 314–424) (see Fig. 1A)) are essential for fibril formation (10, 13). Although all these domains (or fragments thereof) may directly contribute to the fibrillar core (1, 4, 14), the NTR and PKD domains seem to be additionally crucial for subcellular targeting of the polypeptide. Pmel17 lacking either of these domains fails to accumulate in LAMP1/LAMP2-containing organelles when transiently overexpressed in HeLa cells but rather localizes to early endosomes (10, 13). Interestingly, although targeting of Pmel17 to MVBs is normal, its delivery from the limiting membrane to ILVs appears to be impaired when either the NTR or the PKD domain is missing (10). This may underlie the observed subcellular sorting defect of the respective mutants.


Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.

Leonhardt RM, Vigneron N, Rahner C, Van den Eynde BJ, Cresswell P - J. Biol. Chem. (2010)

The NTR is required for normal subcellular targeting of Pmel17. A, schematic representation of the ΔNTR construct. B, ΔNTR can be processed by pPCs. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS/1% β-mercaptoethanol plus protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C, ΔNTR shows both accelerated pPC processing and turnover. Cells from Fig. 1B, were pulse-labeled for 30 min with 35S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS under vigorous vortexing for 30 min, and analyzed by autoradiography (top panel). A 2-day exposure is shown for the upper part of the gel separated by the dashed line from a 5-day exposure for the lower part of the same gel. Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown (bottom panel). D, newly synthesized ΔNTR localizes to the ER and Golgi. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). E, folded HMB50-reactive ΔNTR co-localizes with Golgi and early endosomal markers. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle markers giantin (ab24586) (Golgi) or EEA1 (ab2900) (early endosomes). F, ΔNTR fails to migrate into LAMP1-positive compartments. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle marker LAMP1 (H4A3) (lysosomes/melanosomes).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: The NTR is required for normal subcellular targeting of Pmel17. A, schematic representation of the ΔNTR construct. B, ΔNTR can be processed by pPCs. A total membrane fraction derived from the indicated stable Mel220 transfectants was lysed in 1% SDS/1% β-mercaptoethanol plus protease inhibitors (Complete, Roche Applied Science) and analyzed by Western blot using Pmel17-specific antibodies. C, ΔNTR shows both accelerated pPC processing and turnover. Cells from Fig. 1B, were pulse-labeled for 30 min with 35S and subsequently chased for the indicated times. 2% Triton X-100 lysates were immunoprecipitated with Pmel17-specific antibody HMB50, eluted with 0.5% SDS under vigorous vortexing for 30 min, and analyzed by autoradiography (top panel). A 2-day exposure is shown for the upper part of the gel separated by the dashed line from a 5-day exposure for the lower part of the same gel. Quantitative PhosphorImager analysis of the pulse-chase data with maximal levels for each band set to 100% is shown (bottom panel). D, newly synthesized ΔNTR localizes to the ER and Golgi. Cells from B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). E, folded HMB50-reactive ΔNTR co-localizes with Golgi and early endosomal markers. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle markers giantin (ab24586) (Golgi) or EEA1 (ab2900) (early endosomes). F, ΔNTR fails to migrate into LAMP1-positive compartments. Cells from B were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and organelle marker LAMP1 (H4A3) (lysosomes/melanosomes).
Mentions: The organization of domains within Pmel17 has recently been described, and the first steps have been undertaken to investigate their function in the molecule. In particular, all three large domains within Mα (namely the N-terminal region (NTR, ∼aa 22–210), the polycystic kidney disease-like domain (PKD, ∼aa 211–293), and the repeat domain (RPT, ∼aa 314–424) (see Fig. 1A)) are essential for fibril formation (10, 13). Although all these domains (or fragments thereof) may directly contribute to the fibrillar core (1, 4, 14), the NTR and PKD domains seem to be additionally crucial for subcellular targeting of the polypeptide. Pmel17 lacking either of these domains fails to accumulate in LAMP1/LAMP2-containing organelles when transiently overexpressed in HeLa cells but rather localizes to early endosomes (10, 13). Interestingly, although targeting of Pmel17 to MVBs is normal, its delivery from the limiting membrane to ILVs appears to be impaired when either the NTR or the PKD domain is missing (10). This may underlie the observed subcellular sorting defect of the respective mutants.

Bottom Line: Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment.As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell.Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06519, USA. ralf.leonhardt@yale.edu

ABSTRACT
Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

Show MeSH
Related in: MedlinePlus