scholarly journals Mistargeting of secretory cargo in retromer-deficient cells

2020 ◽  
Author(s):  
Sarah D. Neuman ◽  
Erica L. Terry ◽  
Jane E. Selegue ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Membrane Proteins ◽  
Salivary Glands ◽  
Secretory Granule ◽  
Complex Function ◽  
Secretory Cells ◽  
Regulated Exocytosis ◽  
Retromer Complex ◽  
Cargo Proteins ◽  
Granule Membrane ◽  
Granule Membrane Proteins

ABSTRACTIntracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with Alzheimer’s disease, characterized by accumulation of amyloid β (Aβ). We show that amyloid precursor protein (APP) undergoes regulated exocytosis and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. Our data also suggests that misrouting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.SUMMARY STATEMENTRetromer complex dysfunction is implicated in neurodegeneration. Here the authors show a new role for the retromer complex in recycling of secretory membrane and cargo proteins during regulated exocytosis.

scholarly journals Mistargeting of secretory cargo in retromer-deficient cells

2020 ◽  
pp. dmm.046417
Author(s):  
Sarah D. Neuman ◽  
Erica L. Terry ◽  
Jane E. Selegue ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Membrane Proteins ◽  
Salivary Glands ◽  
Secretory Granule ◽  
Complex Function ◽  
Amyloid Β ◽  
Secretory Cells ◽  
Regulated Exocytosis ◽  
Retromer Complex ◽  
Granule Membrane ◽  
Granule Membrane Proteins

Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). We show that ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.

scholarly journals A novel function for Rab proteins during maturation of secretory granules

2021 ◽  
Author(s):  
Sarah D Neuman ◽  
Annika R Lee ◽  
Jane E Selegue ◽  
Amy T Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Salivary Glands ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Rab Proteins ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Cargo Proteins ◽  
Granule Maturation ◽  
Granule Growth

Regulated exocytosis is an essential process whereby professional secretory cells synthesize and secrete specific cargo proteins in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo many modifications, including a dramatic increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the professional secretory cells of the Drosophila larval salivary glands as a model system to characterize a novel and unexpected role for Rab GTPases during secretory granule maturation. We find that secretory granules in the larval salivary glands increase in size ~300-fold between biogenesis and release, and loss of Rab1 or Rab11 dramatically reduces granule size. Surprisingly, we find that Rab1 and Rab11 protein localize to secretory granule membranes. Rab11 associates with granule membranes throughout the maturation process, and Rab11 is required for recruitment of Rab1. In turn, Rab1 associates specifically with immature secretory granules and drives granule growth. In addition to their roles in granule growth, both Rab1 and Rab11 appear to have additional roles during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new and unexpected role for Rab GTPases in secretory granule maturation.

Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

1990 ◽  
Vol 259 (1) ◽  
pp. G70-G77 ◽  
Author(s):  
C. R. Marino ◽  
J. D. Castle ◽  
F. S. Gorelick
Keyword(s):  
Membrane Proteins ◽  
Parotid Gland ◽  
Secretory Granule ◽  
Integral Membrane Proteins ◽  
Dependent Manner ◽  
Amylase Release ◽  
Rat Parotid Gland ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Granule Membrane Proteins

An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

scholarly journals Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

1983 ◽  
Vol 96 (4) ◽  
pp. 1030-1039 ◽  
Author(s):  
W J Brown ◽  
W A Shannon ◽  
W J Snell
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Granule Protein ◽  
Isolation And Characterization ◽  
Cytoplasmic Face ◽  
Sds Page ◽  
Granule Membrane ◽  
Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

Pancreatic Zymogen Granule Membrane Proteins: Molecular Details Begin to Emerge

Digestion ◽  
1994 ◽  
Vol 55 (4) ◽  
pp. 191-199 ◽  
Author(s):  
Andreas C.C. Wagner ◽  
John A. Williams
Keyword(s):  
Membrane Proteins ◽  
Zymogen Granule ◽  
Granule Membrane ◽  
Granule Membrane Proteins

scholarly journals Atypical parkinsonism–associated retromer mutant alters endosomal sorting of specific cargo proteins

2016 ◽  
Vol 214 (4) ◽  
pp. 389-399 ◽  
Author(s):  
Kirsty J. McMillan ◽  
Matthew Gallon ◽  
Adam P. Jellett ◽  
Thomas Clairfeuille ◽  
Frances C. Tilley ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Neurodegenerative Disease ◽  
Quantitative Proteomics ◽  
Protein Complexes ◽  
Integral Membrane Proteins ◽  
Atypical Parkinsonism ◽  
Endosomal Sorting ◽  
Retromer Complex ◽  
Cargo Proteins ◽  
Cargo Sorting

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism.

TOPOLOGY ANALYSIS OF PANCREATIC ZYMOGEN GRANULE MEMBRANE PROTEINS

Pancreas ◽  
2007 ◽  
pp. 396 ◽  
Author(s):  
X. Chen ◽  
E. S. Simon ◽  
P. C. Andrews ◽  
J. A. Williams
Keyword(s):  
Membrane Proteins ◽  
Zymogen Granule ◽  
Topology Analysis ◽  
Granule Membrane ◽  
Granule Membrane Proteins
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