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Selective requirement of a deacetylase domain for Emp24independent luminal secretion in the Drosophila trachea
Stockholm University, Faculty of Science, The Wenner-Gren Institute . (Christos Samakovlis)
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Vermiform and Serpentine are secreted putative chitin deacetylases (ChLDs). They are deposited into the tracheal lumen to terminate tube elongation during morphogenesis. Deletion analysis of a Serp-GFP reporter had revealed that the deacetylase domain is essential for its luminal localization. We transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The GaspDeac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To explore this possibility we identified and characterized conserved sequence motifs in Serp deacetylase domain. We generated amino acid substitution mutants altering the three putative Nglycosylation sites, the predicted enzymatic activity cluster and three phylogenetically conserved motifs. We tested the cellular localization of the constructs in S2 cultured cells and the trachea of transgenic Drosophila embryos. Residue substitutions in the putative catalytic site neither interfered with Serp secretion nor with its ability to rescue the tracheal tube elongation defects of serp mutants. Mutations of the N-glycosylation sites gradually reduced the luminal deposition of Serp-GFP constructs suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in each of the three uncharacterized amino acid stretches completely blocked the ER-exit of Serp-GFP constructs. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors.

Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-32570OAI: oai:DiVA.org:su-32570DiVA: diva2:280975
Available from: 2009-12-15 Created: 2009-12-14 Last updated: 2011-05-06Bibliographically approved
In thesis
1. New roles for apical secretion and extracellular matrix assembly in Drosophila epithelial morphogenesis
Open this publication in new window or tab >>New roles for apical secretion and extracellular matrix assembly in Drosophila epithelial morphogenesis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Branched tubular organs, such as the lung and vascular system fulfill the respiratory needs of most animals. Optimal tissue function relies on the uniform sizes and shapes of the constituting branches in each organ. The Drosophila tracheal airways provide a recognized genetic model system for identification and characterization of tube size regulators. We found that the programmed secretion and assembly of the apical extracellular matrix (ECM) is required for the expansion of the trachea and salivary glands (SG) tubes. We have characterized Vermiform (Verm) and Serpentine (Serp), two chitin-binding proteins with predicted polysaccharide deacetylase domains (ChLDs). Verm and Serp mutants show overelongated tubes, suggesting that luminal ECM modification restricts tracheal tube elongation. The luminal deposition of ChLDs, but not other secreted components, depends on paracellular septate junction integrity (SJs) in the tracheal epithelium. Deletion of the deacetylase domain renders Serp-GFP intracellular, arguing that the deacetylase domain harbors uncharacterized secretion signals. To explore this possibility we transferred the deacetylase domain from Serp to Gasp, another tracheal luminal protein, which requires the Emp24 adaptor for ER exit. The Gasp-Deac-GFP chimera was normally secreted in emp24 mutants indicating that the deacetylase domain contains potential ER-exit signals. To identify such signals we characterized conserved sequence motifs in the Serp deacetylase domain. Mutations of the N-glycosylation sites gradually reduced Serp-GFP luminal deposition suggesting that increased glycosylation enhances apical Serp secretion. By contrast, substitutions in three conserved amino acid stretches completely blocked the ER-exit of Serp-GFP. The mutated proteins were N-glycosylated suggesting that the motifs may be involved in a subsequent protein-folding step or facilitate ER exit through interactions with unidentified specific adaptors.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2010. 64 p.
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-32564 (URN)978-91-7155-952-4 (ISBN)
Public defence
2010-01-29, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript. Available from: 2010-01-07 Created: 2009-12-14 Last updated: 2011-02-28Bibliographically approved

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