Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells
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S L Milgram1, S T Kho, G V Martin
1Physiology Department, The University of North Carolina at Chapel Hill 27599, USA.
Abstract:
Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.
Insights
Peptidylglycine alpha-amidating monooxygenase (PAM) localizes to perinuclear tubulovesicular structures in neuroendocrine cells. This membrane protein is found in the trans-Golgi network but not primarily in endosomes or lysosomes.
Area of Science:
- Cell Biology
- Neuroendocrinology
- Protein Trafficking
Background:
- Peptidylglycine alpha-amidating monooxygenase (PAM) is crucial for peptide hormone amidation in neuroendocrine cells.
- Membrane-bound PAM accumulates in a perinuclear region at steady state.
- Understanding PAM localization is key to elucidating neuroendocrine secretion pathways.
Purpose of the Study:
- To determine the precise subcellular localization of membrane PAM in neuroendocrine cells.
- To compare PAM distribution with markers of the trans-Golgi network (TGN), endosomes, and lysosomes.
- To investigate the relationship between PAM localization and the trafficking of POMC-derived peptides.
Main Methods:
- Stable transfection of AtT-20 cells with membrane PAM.
- Light and immunoelectron microscopy using specific cellular markers (TGN38, SCAMP, lysosomal markers, FITC-transferrin, HRP, WGA-HRP).
- Analysis of co-localization with POMC-derived peptides and internalized tracers.
Main Results:
- Membrane PAM co-localizes with TGN38 and SCAMP, indicating presence in the TGN and post-Golgi recycling pathways.
- PAM is found in tubulovesicular structures within the TGN, some distal to Golgi stacks and lacking TGN38.
- While some POMC peptides are in PAM-containing structures, most reside in secretory granules; minimal overlap with early endosomes.
Conclusions:
- Membrane PAM is primarily localized to perinuclear tubulovesicular structures associated with the TGN.
- These PAM-containing compartments are partially distinct from TGN38 and are not exclusively endosomal.
- This localization suggests a specific role for these TGN-derived structures in PAM trafficking and function.