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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 integrity of a small region in the vicinity of the NTR-PKD domain boundary is crucial for ER exit of Pmel17. A, schematic representation of the Δ190–208 construct. B, only the ER-associated P1 form can be detected for Δ190–208 at steady state. Membrane lysates of Mel220 transfectants stably expressing Δ190–208 were prepared as in Fig. 1B and analyzed by Western blot using Pmel17-specific antibodies. C, newly synthesized Δ190–208 is completely retained in the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). D, folded NKI-beteb-reactive Δ190–208 is completely absent from the cell surface. Cells from Fig. 2B were surface-labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry (left panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (right panel). E, low levels of folded Δ190–208 can be detected intracellularly. Cells from Fig. 2B were fixed in 2% formaldehyde, permeabilized, and stained intracellularly with antibodies reactive with folded (NKI-beteb or HMB50) or newly synthesized Pmel17 (Pep13h) and analyzed by flow cytometry (top panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (bottom panel). F, folded HMB50- and NKI-beteb-reactive Δ190–208 localizes to the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against folded Pmel17 (NKI-beteb (top panel) or HMB50 (bottom panel)) and newly synthesized Pmel17 (Pmel-N (top panel) or Pep13h (bottom panel)). G, Δ190–208 does not react with antibody HMB45, which specifically recognizes sialylated (post-Golgi- or Golgi-localized) Pmel17. Cells from Fig. 2B were treated or not with 10 μg/ml brefeldin A (BFA) for 4 h to shift Golgi-associated proteins back into the ER and analyzed by immunofluorescence using antibodies against sialylated (post-Golgi or Golgi-localized) Pmel17 (HMB45) and organelle marker giantin (ab24586) (Golgi).
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Figure 2: The integrity of a small region in the vicinity of the NTR-PKD domain boundary is crucial for ER exit of Pmel17. A, schematic representation of the Δ190–208 construct. B, only the ER-associated P1 form can be detected for Δ190–208 at steady state. Membrane lysates of Mel220 transfectants stably expressing Δ190–208 were prepared as in Fig. 1B and analyzed by Western blot using Pmel17-specific antibodies. C, newly synthesized Δ190–208 is completely retained in the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). D, folded NKI-beteb-reactive Δ190–208 is completely absent from the cell surface. Cells from Fig. 2B were surface-labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry (left panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (right panel). E, low levels of folded Δ190–208 can be detected intracellularly. Cells from Fig. 2B were fixed in 2% formaldehyde, permeabilized, and stained intracellularly with antibodies reactive with folded (NKI-beteb or HMB50) or newly synthesized Pmel17 (Pep13h) and analyzed by flow cytometry (top panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (bottom panel). F, folded HMB50- and NKI-beteb-reactive Δ190–208 localizes to the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against folded Pmel17 (NKI-beteb (top panel) or HMB50 (bottom panel)) and newly synthesized Pmel17 (Pmel-N (top panel) or Pep13h (bottom panel)). G, Δ190–208 does not react with antibody HMB45, which specifically recognizes sialylated (post-Golgi- or Golgi-localized) Pmel17. Cells from Fig. 2B were treated or not with 10 μg/ml brefeldin A (BFA) for 4 h to shift Golgi-associated proteins back into the ER and analyzed by immunofluorescence using antibodies against sialylated (post-Golgi or Golgi-localized) Pmel17 (HMB45) and organelle marker giantin (ab24586) (Golgi).

Mentions: Our data (Fig. 1, E and F) and the data of others (10, 13) demonstrate that the presence of the NTR is essential for normal trafficking of Pmel17. We next wanted to assess whether a smaller subregion within the N terminus could be identified that is required for normal subcellular distribution of the protein. To this end, we introduced an 18-aa deletion into Pmel17 comprising only residues 190–208 (Fig. 2A) (hereinafter Δ190–208). Thus, Δ190–208 and ΔNTR differ only by the N-terminal extension of the respective deletion but share its C-terminal border. This border is located only 2 aa before the junction of exons 6 and 7. We note that exon boundaries are well known to correlate with domain boundaries in many polypeptides (32), and within Pmel17 the respective area likely corresponds to the border between the NTR and the PKD domains (Fig. 2A). Further supporting this, domain linker prediction using domain linker prediction-support vector machine (DLP-SVM) (33) identifies region 200–217 within a Pmel17 sample sequence ranging from aa 160 to 280 as the most likely area to represent a loop separating two independent structural domains.


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 integrity of a small region in the vicinity of the NTR-PKD domain boundary is crucial for ER exit of Pmel17. A, schematic representation of the Δ190–208 construct. B, only the ER-associated P1 form can be detected for Δ190–208 at steady state. Membrane lysates of Mel220 transfectants stably expressing Δ190–208 were prepared as in Fig. 1B and analyzed by Western blot using Pmel17-specific antibodies. C, newly synthesized Δ190–208 is completely retained in the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). D, folded NKI-beteb-reactive Δ190–208 is completely absent from the cell surface. Cells from Fig. 2B were surface-labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry (left panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (right panel). E, low levels of folded Δ190–208 can be detected intracellularly. Cells from Fig. 2B were fixed in 2% formaldehyde, permeabilized, and stained intracellularly with antibodies reactive with folded (NKI-beteb or HMB50) or newly synthesized Pmel17 (Pep13h) and analyzed by flow cytometry (top panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (bottom panel). F, folded HMB50- and NKI-beteb-reactive Δ190–208 localizes to the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against folded Pmel17 (NKI-beteb (top panel) or HMB50 (bottom panel)) and newly synthesized Pmel17 (Pmel-N (top panel) or Pep13h (bottom panel)). G, Δ190–208 does not react with antibody HMB45, which specifically recognizes sialylated (post-Golgi- or Golgi-localized) Pmel17. Cells from Fig. 2B were treated or not with 10 μg/ml brefeldin A (BFA) for 4 h to shift Golgi-associated proteins back into the ER and analyzed by immunofluorescence using antibodies against sialylated (post-Golgi or Golgi-localized) Pmel17 (HMB45) and organelle marker giantin (ab24586) (Golgi).
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Figure 2: The integrity of a small region in the vicinity of the NTR-PKD domain boundary is crucial for ER exit of Pmel17. A, schematic representation of the Δ190–208 construct. B, only the ER-associated P1 form can be detected for Δ190–208 at steady state. Membrane lysates of Mel220 transfectants stably expressing Δ190–208 were prepared as in Fig. 1B and analyzed by Western blot using Pmel17-specific antibodies. C, newly synthesized Δ190–208 is completely retained in the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against newly synthesized Pmel17 (Pep13h) and organelle markers TAP1 (148.3) (ER) or GM130 (610823) (Golgi). D, folded NKI-beteb-reactive Δ190–208 is completely absent from the cell surface. Cells from Fig. 2B were surface-labeled with antibody NKI-beteb against folded Pmel17 and analyzed by flow cytometry (left panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (right panel). E, low levels of folded Δ190–208 can be detected intracellularly. Cells from Fig. 2B were fixed in 2% formaldehyde, permeabilized, and stained intracellularly with antibodies reactive with folded (NKI-beteb or HMB50) or newly synthesized Pmel17 (Pep13h) and analyzed by flow cytometry (top panel). After background subtraction (untransfected Mel220 cells) data are represented as a bar diagram (bottom panel). F, folded HMB50- and NKI-beteb-reactive Δ190–208 localizes to the ER. Cells from Fig. 2B were analyzed by immunofluorescence using antibodies against folded Pmel17 (NKI-beteb (top panel) or HMB50 (bottom panel)) and newly synthesized Pmel17 (Pmel-N (top panel) or Pep13h (bottom panel)). G, Δ190–208 does not react with antibody HMB45, which specifically recognizes sialylated (post-Golgi- or Golgi-localized) Pmel17. Cells from Fig. 2B were treated or not with 10 μg/ml brefeldin A (BFA) for 4 h to shift Golgi-associated proteins back into the ER and analyzed by immunofluorescence using antibodies against sialylated (post-Golgi or Golgi-localized) Pmel17 (HMB45) and organelle marker giantin (ab24586) (Golgi).
Mentions: Our data (Fig. 1, E and F) and the data of others (10, 13) demonstrate that the presence of the NTR is essential for normal trafficking of Pmel17. We next wanted to assess whether a smaller subregion within the N terminus could be identified that is required for normal subcellular distribution of the protein. To this end, we introduced an 18-aa deletion into Pmel17 comprising only residues 190–208 (Fig. 2A) (hereinafter Δ190–208). Thus, Δ190–208 and ΔNTR differ only by the N-terminal extension of the respective deletion but share its C-terminal border. This border is located only 2 aa before the junction of exons 6 and 7. We note that exon boundaries are well known to correlate with domain boundaries in many polypeptides (32), and within Pmel17 the respective area likely corresponds to the border between the NTR and the PKD domains (Fig. 2A). Further supporting this, domain linker prediction using domain linker prediction-support vector machine (DLP-SVM) (33) identifies region 200–217 within a Pmel17 sample sequence ranging from aa 160 to 280 as the most likely area to represent a loop separating two independent structural domains.

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