Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

Bonnemaison, Mathilde L.; Back, Nathan; Duffy, Megan; Ralle, Martina; Mains, Richard E.; Eipper, Betty A.

doi:10.1074/jbc.m115.641027

Cited by 12 publications

(18 citation statements)

References 60 publications

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“…Similar to previous results from our laboratory using a VWF-expressing HEK293 cell model, 17 downregulation of AP-1m1A in HUVECs impairs WPB formation (supplemental Figure 2), with a resulting increase in unstimulated VWF secretion ( Figure 4C, nonstriped bars) plus a decrease in stimulated VWF release ( Figure 4D), when cells were grown in plastic wells. Critically, this was not caused by Golgi fragmentation in siAP-1M1-treated cells, 27,28 (supplemental Figure 2) because we know this would affect WPB biogenesis. 29 Using BFA, we can also now show that most of this increase in unstimulated VWF release in siAP-1M1-treated cells is caused by an increase in secretion via the constitutive pathway, because a larger proportion was blocked with BFA ( Figure 4C).…”

Section: Ap-1 and Vwf Polar Targetingmentioning

confidence: 99%

von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

Silva

Cutler

2016

Blood

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Key Points• The 3 endothelial secretory pathways-constitutive, basal, and regulated-release VWF in different multimeric states.• Apical-and basolaterallyreleased VWF follow different secretory pathways, thus releasing differentially multimerized protein.The von Willebrand factor (VWF) synthesized and secreted by endothelial cells is central to hemostasis and thrombosis, providing a multifunctional adhesive platform that brings together components needed for these processes. VWF secretion can occur from both apical and basolateral sides of endothelial cells, and from constitutive, basal, and regulated secretory pathways, the latter two via Weibel-Palade bodies (WPB). Although the amount and structure of VWF is crucial to its function, the extent of VWF release, multimerization, and polarity of the 3 secretory pathways have only been addressed separately, and with conflicting results. We set out to clarify these relationships using polarized human umbilical vein endothelial cells (HUVECs) grown on Transwell membranes. We found that regulated secretion of ultra-large (UL)-molecular-weight VWF predominantly occurred apically, consistent with a role in localized platelet capture in the vessel lumen. We found that constitutive secretion of low-molecular-weight (LMW) VWF is targeted basolaterally, toward the subendothelial matrix, using the adaptor protein complex 1 (AP-1), where it may provide the bulk of collagen-bound subendothelial VWF. We also found that basally-secreted VWF is composed of UL-VWF, released continuously from WPBs in the absence of stimuli, and occurs predominantly apically, suggesting this could be the main source of circulating plasma VWF. Together, we provide a unified dataset reporting the amount and multimeric state of VWF secreted from the constitutive, basal, and regulated pathways in polarized HUVECs, and have established a new role for AP-1 in the basolateral constitutive secretion of VWF. (Blood. 2016;128(2):277-285)

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Section: Ap-1 and Vwf Polar Targetingmentioning

confidence: 99%

von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

Silva

Cutler

2016

Blood

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“…PAM, which reaches the plasma membrane following exocytosis, is rapidly internalized and returns to the TGN through pathways involving its regulated entry into multivesicular bodies and then into intraluminal vesicles (Bäck, Rajagopal, Mains, & Eipper, 2010) After blue native PAGE followed by SDS-PAGE, PAM-1 was found in a horizontal streak, indicating its presence in multiple complexes ranging in mass from 300 to 1000 kDa. PAM was previously shown to interact with a number of secretory and endocytic proteins along with several cytosolic proteins (Bonnemaison et al, 2015;Chen, Johnson, & Milgram, 1998;Francone et al, 2010;Mains et al, 1999;Otoikhian et al, 2012). Results from two-dimensional gels and mass spectroscopy analysis confirm the presence of all of the core V-ATPase subunits in a footprint along with PAM.…”

Section: Pam and V-atpase Coexist On Endomembranes Of Peptidergic Cmentioning

confidence: 63%

A pH‐sensitive luminal His‐cluster promotes interaction of PAM with V‐ATPase along the secretory and endocytic pathways of peptidergic cells

Rao

Zavala

Roy

et al. 2018

Journal Cellular Physiology

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The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar‐type‐H+ ATPase (V‐ATPase). V‐ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V‐ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α‐amidating mono‐oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V‐ATPase accessory proteins (Ac45 and prorenin receptor), has a pH‐sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT‐20) identified distinct subcellular compartments exhibiting spatial co‐occurrence of PAM and V‐ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high‐molecular weight complexes of PAM and V‐ATPase. A PAM‐1 mutant (PAM‐1/H3A) with altered pH sensitivity had diminished ability to form high‐molecular weight complexes. In addition, V‐ATPase assembly status was altered in PAM‐1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH‐sensor, regulating V‐ATPase action by altering its assembly status.

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“…Uhmk1, a Ser/Thr kinase, phosphorylates the C-terminus of PAM and Rassf9, a member of the Ras-association domain family, plays a role in epidermal homeostasis (Alam, et al 1996; Lee, et al 2011). More recently, PAM has been shown to interact with the Adaptor Protein-1 (AP-1) Complex (Bonnemaison, et al 2015). The AP-1 complex belongs to a family of endosomal and secretory granule coat proteins that link cargo proteins to clathrin; both PAM and Atp7a, the P-type ATPase that transports copper into the TGN, interact with AP-1.…”

Section: More Than Just An Enzyme - a Multi-tasking Proteinmentioning

confidence: 99%

“…Diminished AP-1 function affects PAM and Atp7a trafficking through the endocytic pathway. Antibodies to amidated (18 kDa fragment) and non-amidated POMC products allowed development of an assay to measure PAM activity in cells following manipulation of copper levels; cells with reduced AP-1 levels were more sensitive to copper restriction than control cells (Bonnemaison, et al 2014; Bonnemaison et al 2015). …”

Section: More Than Just An Enzyme - a Multi-tasking Proteinmentioning

confidence: 99%

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C

Kumar

Mains

Eipper

2016

Journal of Molecular Endocrinology

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A critical role for peptide C-terminal amidation was apparent when the first bioactive peptides were identified. The conversion of POMC into ACTH and then into αMSH, an amidated peptide, provided a model system for identifying the amidating enzyme. Peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that catalyzes this modification, is essential; mice lacking PAM survive only until mid-gestation. Purification and cloning led to the discovery that the amidation of peptidylglycine substrates proceeds in two steps: peptidylglycine α-hydroxylating monooxygenase (PHM) catalyzes the copper and ascorbate-dependent α-hydroxylation of the peptidylglycine substrate; peptidyl-α-hydroxyglycine α-amidating lyase (PAL) cleaves the N-C bond, producing amidated product plus glyoxylate. Both enzymes are contained in the luminal domain of PAM, a type 1 integral membrane protein. The structures of both catalytic cores have been determined, revealing how they interact with metals, molecular oxygen and substrate to catalyze both reactions. Although not essential for activity, the intrinsically disordered cytosolic domain is essential for PAM trafficking. A phylogenetic survey led to identification of bifunctional membrane PAM in Chlamydomonas, a unicellular eukaryote. Accumulating evidence points to a role for PAM in copper homeostasis and in retrograde signaling from the lumen of the secretory pathway to the nucleus. The discovery of PAM in cilia, cellular antennae that sense and respond to environmental stimuli, suggests that much remains to be learned about this ancient protein.

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Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme

Cited by 12 publications

References 60 publications

von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

A pH‐sensitive luminal His‐cluster promotes interaction of PAM with V‐ATPase along the secretory and endocytic pathways of peptidergic cells

60 YEARS OF POMC: From POMC and α-MSH to PAM, molecular oxygen, copper, and vitamin C

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