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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 post-ER populations of IR-wt and H190P are non-functional and largely route to lysosomes. A, IR-wt is severely impaired or blocked in fibril formation. Electron microscopic analysis of Epon-embedded Mel220 transfectants stably expressing wt-Pmel17 or IR-wt. Panel 1 shows a typical melanosome frequently found for wt-Pmel17. Panel 2 shows an example of the very rare occurrence of immature organelles that contain individual striae-like structures (white arrowheads). Almost always such structures could be clearly identified as membrane segments as judged by a visible double layer structure. However, in a few remaining cases image quality did not allow us to fully exclude that they represent an immature fibril in the formation process. Hence, IR-wt is at least severely impaired, but more likely completely blocked in fibril formation. B, folded HMB50-reactive IR-wt gets mostly delivered to compartments with lysosomal morphology. Cells expressing wt-Pmel17 or IR-wt were fixed and examined by cryo-immunoelectron microscopy (panels 1–5) using antibody HMB50. Panels 1 and 2 display MVBs with extensive immunolabeling over intralumenal vesicles. Panel 3 shows a typical melanosome in wt-Pmel17-expressing cells. Black arrowheads in panels 4 and 5 point to lysosomal, often multilamellar compartments densely labeled with gold particles in IR-wt-expressing cells. C, wt-Pmel17 mostly distributes to LAMP1low melanosomes, whereas IR-wt and H190P extensively co-localize with LAMP1 in LAMP1high lysosomes. Mel220 cells stably expressing the indicated Pmel17 mutants were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and LAMP1 (H4A3). D, quantification of the average difference (see “Experimental Procedures”) between the Pmel17 and the LAMP1 profiles in the indicated Mel220 transfectants in a statistically relevant number of cells. The pattern differences between wt-Pmel17 and either IR-wt (*, p < 0.05) or H190P (**, p < 0.01) are statistically significant, as assessed by a one-way analysis of variance test with the Dunnett post-test. Pattern differences between IR-wt and H190P are not statistically significant (NS).
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Figure 5: The post-ER populations of IR-wt and H190P are non-functional and largely route to lysosomes. A, IR-wt is severely impaired or blocked in fibril formation. Electron microscopic analysis of Epon-embedded Mel220 transfectants stably expressing wt-Pmel17 or IR-wt. Panel 1 shows a typical melanosome frequently found for wt-Pmel17. Panel 2 shows an example of the very rare occurrence of immature organelles that contain individual striae-like structures (white arrowheads). Almost always such structures could be clearly identified as membrane segments as judged by a visible double layer structure. However, in a few remaining cases image quality did not allow us to fully exclude that they represent an immature fibril in the formation process. Hence, IR-wt is at least severely impaired, but more likely completely blocked in fibril formation. B, folded HMB50-reactive IR-wt gets mostly delivered to compartments with lysosomal morphology. Cells expressing wt-Pmel17 or IR-wt were fixed and examined by cryo-immunoelectron microscopy (panels 1–5) using antibody HMB50. Panels 1 and 2 display MVBs with extensive immunolabeling over intralumenal vesicles. Panel 3 shows a typical melanosome in wt-Pmel17-expressing cells. Black arrowheads in panels 4 and 5 point to lysosomal, often multilamellar compartments densely labeled with gold particles in IR-wt-expressing cells. C, wt-Pmel17 mostly distributes to LAMP1low melanosomes, whereas IR-wt and H190P extensively co-localize with LAMP1 in LAMP1high lysosomes. Mel220 cells stably expressing the indicated Pmel17 mutants were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and LAMP1 (H4A3). D, quantification of the average difference (see “Experimental Procedures”) between the Pmel17 and the LAMP1 profiles in the indicated Mel220 transfectants in a statistically relevant number of cells. The pattern differences between wt-Pmel17 and either IR-wt (*, p < 0.05) or H190P (**, p < 0.01) are statistically significant, as assessed by a one-way analysis of variance test with the Dunnett post-test. Pattern differences between IR-wt and H190P are not statistically significant (NS).

Mentions: However, we noted that this condensed perinuclear pattern typical of NKI-beteb-/HMB50-/HMB45-reactive IR-wt differed substantially from the subcellular pattern detected for wt-Pmel17 (Fig. 4B). In particular, wt-Pmel17 seemed to distribute into a broad band surrounding the perinuclear area and resembling a “horseshoe profile” (and in some cases of very high expression Pmel17 was scattered all over the cell) (Fig. 4, B and D; see also Fig. 5C). This suggests that, although both wt-Pmel17 and IR-wt efficiently label with conformation-sensitive antibodies, their respective post-ER populations reside in different locations in the cell.


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 post-ER populations of IR-wt and H190P are non-functional and largely route to lysosomes. A, IR-wt is severely impaired or blocked in fibril formation. Electron microscopic analysis of Epon-embedded Mel220 transfectants stably expressing wt-Pmel17 or IR-wt. Panel 1 shows a typical melanosome frequently found for wt-Pmel17. Panel 2 shows an example of the very rare occurrence of immature organelles that contain individual striae-like structures (white arrowheads). Almost always such structures could be clearly identified as membrane segments as judged by a visible double layer structure. However, in a few remaining cases image quality did not allow us to fully exclude that they represent an immature fibril in the formation process. Hence, IR-wt is at least severely impaired, but more likely completely blocked in fibril formation. B, folded HMB50-reactive IR-wt gets mostly delivered to compartments with lysosomal morphology. Cells expressing wt-Pmel17 or IR-wt were fixed and examined by cryo-immunoelectron microscopy (panels 1–5) using antibody HMB50. Panels 1 and 2 display MVBs with extensive immunolabeling over intralumenal vesicles. Panel 3 shows a typical melanosome in wt-Pmel17-expressing cells. Black arrowheads in panels 4 and 5 point to lysosomal, often multilamellar compartments densely labeled with gold particles in IR-wt-expressing cells. C, wt-Pmel17 mostly distributes to LAMP1low melanosomes, whereas IR-wt and H190P extensively co-localize with LAMP1 in LAMP1high lysosomes. Mel220 cells stably expressing the indicated Pmel17 mutants were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and LAMP1 (H4A3). D, quantification of the average difference (see “Experimental Procedures”) between the Pmel17 and the LAMP1 profiles in the indicated Mel220 transfectants in a statistically relevant number of cells. The pattern differences between wt-Pmel17 and either IR-wt (*, p < 0.05) or H190P (**, p < 0.01) are statistically significant, as assessed by a one-way analysis of variance test with the Dunnett post-test. Pattern differences between IR-wt and H190P are not statistically significant (NS).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: The post-ER populations of IR-wt and H190P are non-functional and largely route to lysosomes. A, IR-wt is severely impaired or blocked in fibril formation. Electron microscopic analysis of Epon-embedded Mel220 transfectants stably expressing wt-Pmel17 or IR-wt. Panel 1 shows a typical melanosome frequently found for wt-Pmel17. Panel 2 shows an example of the very rare occurrence of immature organelles that contain individual striae-like structures (white arrowheads). Almost always such structures could be clearly identified as membrane segments as judged by a visible double layer structure. However, in a few remaining cases image quality did not allow us to fully exclude that they represent an immature fibril in the formation process. Hence, IR-wt is at least severely impaired, but more likely completely blocked in fibril formation. B, folded HMB50-reactive IR-wt gets mostly delivered to compartments with lysosomal morphology. Cells expressing wt-Pmel17 or IR-wt were fixed and examined by cryo-immunoelectron microscopy (panels 1–5) using antibody HMB50. Panels 1 and 2 display MVBs with extensive immunolabeling over intralumenal vesicles. Panel 3 shows a typical melanosome in wt-Pmel17-expressing cells. Black arrowheads in panels 4 and 5 point to lysosomal, often multilamellar compartments densely labeled with gold particles in IR-wt-expressing cells. C, wt-Pmel17 mostly distributes to LAMP1low melanosomes, whereas IR-wt and H190P extensively co-localize with LAMP1 in LAMP1high lysosomes. Mel220 cells stably expressing the indicated Pmel17 mutants were analyzed by immunofluorescence using antibodies against folded Pmel17 (HMB50) and LAMP1 (H4A3). D, quantification of the average difference (see “Experimental Procedures”) between the Pmel17 and the LAMP1 profiles in the indicated Mel220 transfectants in a statistically relevant number of cells. The pattern differences between wt-Pmel17 and either IR-wt (*, p < 0.05) or H190P (**, p < 0.01) are statistically significant, as assessed by a one-way analysis of variance test with the Dunnett post-test. Pattern differences between IR-wt and H190P are not statistically significant (NS).
Mentions: However, we noted that this condensed perinuclear pattern typical of NKI-beteb-/HMB50-/HMB45-reactive IR-wt differed substantially from the subcellular pattern detected for wt-Pmel17 (Fig. 4B). In particular, wt-Pmel17 seemed to distribute into a broad band surrounding the perinuclear area and resembling a “horseshoe profile” (and in some cases of very high expression Pmel17 was scattered all over the cell) (Fig. 4, B and D; see also Fig. 5C). This suggests that, although both wt-Pmel17 and IR-wt efficiently label with conformation-sensitive antibodies, their respective post-ER populations reside in different locations in the cell.

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