AP-3 Mediates Tyrosinase but Not TRP-1 Trafficking in Human Melanocytes

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the β3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the β3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3–deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

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Genetically encoded multimode reporter of adaptor protein 3 (AP-3) traffic in budding yeast

10.1101/314286 ◽

2018 ◽

Author(s):

Rachael L. Plemel ◽

Greg Odorizzi ◽

Alexey J. Merz

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Budding Yeast ◽

Agar Plate ◽

Adaptor Protein ◽

Epileptic Encephalopathy ◽

Early Endosomes ◽

Hermansky Pudlak Syndrome ◽

Functional Deficiency

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening.SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathway’s importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

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A Role of Phosphatidylserine in the Function of Recycling Endosomes

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.783857 ◽

2021 ◽

Vol 9 ◽

Author(s):

Junya Hasegawa ◽

Yasunori Uchida ◽

Kojiro Mukai ◽

Shoken Lee ◽

Tatsuyuki Matsudaira ◽

...

Keyword(s):

Cell Signalling ◽

Point Of View ◽

Syndrome Type ◽

Recycling Endosomes ◽

Emerging Issues ◽

Hermansky Pudlak Syndrome ◽

Recycling Pathway ◽

Established Role

Cells internalize proteins and lipids in the plasma membrane (PM) and solutes in the extracellular space by endocytosis. The removal of PM by endocytosis is constantly balanced by the replenishment of proteins and lipids to PM through recycling pathway. Recycling endosomes (REs) are specific subsets of endosomes. Besides the established role of REs in recycling pathway, recent studies have revealed unanticipated roles of REs in membrane traffic and cell signalling. In this review, we highlight these emerging issues, with a particular focus on phosphatidylserine (PS), a phospholipid that is highly enriched in the cytosolic leaflet of RE membranes. We also discuss the pathogenesis of Hermansky Pudlak syndrome type 2 (HPS2) that arises from mutations in the AP3B1 gene, from the point of view of dysregulated RE functions.

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A pro-inflammatory role of type 2 innate lymphoid cells in murine immune-mediated hepatitis

Zeitschrift für Gastroenterologie ◽

10.1055/s-0035-1567976 ◽

2015 ◽

Vol 53 (12) ◽

Author(s):

K Karimi ◽

K Neumann ◽

J Meiners ◽

R Voetlause ◽

W Dammermann ◽

...

Keyword(s):

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Immune Mediated

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Relationship Between Mepacrine-Labelled Dense Body Number, Platelet Capacity to Accumulate 14C-5-HT and Platelet Density in the Bernard-Soulier and Hermansky-Pudlak Syndromes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666908 ◽

1979 ◽

Vol 42 (02) ◽

pp. 694-704 ◽

Cited By ~ 31

Author(s):

F Rendu ◽

A T Nurden ◽

M Lebret ◽

J P Caen

Keyword(s):

Dense Body ◽

Human Subjects ◽

Human Platelets ◽

Dense Bodies ◽

Storage Organelle ◽

Hereditary Disorders ◽

Labelling Procedure ◽

Normal Human ◽

Hermansky Pudlak Syndrome ◽

Bernard Soulier Syndrome

SummaryWe have used the mepacrine-labelling procedure to measure the dense body (serotonin storage organelle) content of the platelets of 2 hereditary disorders where abnormalities in dense body number were suspected. The platelets were incubated with mepacrine and examined by fluorescence microscopy. A mean number of 5.4 ± 0.8 (SD) dense bodies per platelet was calculated from the data obtained using platelets isolated from 40 normal human subjects. In contrast the platelets of 2 patients with the Bernard-Soulier syndrome contained an average of 14 and 17 labelled granules. This increase was associated with a much greater capacity of the platelets to accumulate 14C-5-HT. The opposite result was obtained using the platelets from 2 patients with the Hermansky-Pudlak syndrome which contained few granules labelled by mepacrine and took up less 14C-5-HT than normal human platelets. Centrifugation of the patients’ platelets on discontinuous sucrose gradients showed that the platelets of the 2 Bemard-Soulier patients were much denser than normal whereas a high proportion of low density platelets was observed in the Hermansky-Pudlak syndrome. These results further define the platelet abnormalities in the two syndromes and suggest that dense body number may be one of the factors governing platelet density.

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