Transport and stability of the Drosophila photoreceptor proteins rhodopsin and TRPL
Final Report Abstract
Intracellular trafficking of membrane proteins is of fundamental importance for cell function and cell integrity. In order to maintain proteostasis, trafficking processes resulting in supply of the plasma membrane with newly synthesized proteins, internalization, recycling and degradation are precisely balanced between the secretory pathway and components of the endolysosomal network. Defects in protein trafficking disturb proteostasis and may result in neuronal degeneration causing blinding diseases such as retinitis pigmentosa as well as other neuronal disorders. The compound eye of Drosophila has emerged as a model system for studying mechanisms of protein trafficking and retinal degeneration in vivo. Regulated trafficking of the rhabdomeric membrane proteins Rh1 (rhodopsin1) and TRPL (transient receptor potential like) within the endosomal network is essential for the proper function of the visual system. Upon a light stimulus, TRPL and Rh1 are internalized from the rhabdomeric membrane to the cell body by endocytotic vesicles. Whereas the majority of internalized rhodopsin is directed to the lysosome for degradation, most of the internalized TRPL is destined for endosomal recycling. In the project we have focused on mechanisms and molecular components underlying transport and recycling of TRPL. By photoreceptor specific expression of TRPL::eGFP and a newly generated fusion protein, TRPL::SNAP, which allows self-labeling with fluorophores, we investigated problems with accurate immunocytochemical detection of rhabdomeric proteins. Our results showed that a frequently observed crescent shaped staining pattern for rhodopsin, TRP and TRPL at the rim of the rhabdomeres is a labeling artifact. TRPL::SNAP co-immunoprecipitation approaches identified potential cellular interaction partners of TRPL, including, among others, the endoplasmic reticulum (ER) membrane protein SERCA. By detecting Co-localization of TRPL and organelle markers we found that TRPL is preferentially located in early and late endosomes after 2 hours of illumination. After 16 hours of illumination co-localization of TRPL and the ER marker protein calnexin was observed. This finding suggests an unexpected recycling route involving the ER aside from the commonly described recycling of membrane proteins via recycling endosome or the trans-Golgi network. A previously described orphan protein, termed TTD14, is required for TRPL recycling, which is inhibited in the mutant ttd14P75L. We showed that TTD14, but not the mutant TTD14P75L, binds GTP and exhibits low intrinsic GTPase activity when expressed in eukaryotic SF9 cells. Both, TTD14 and TTD14P75L bind to the phospholipids phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidic acid (PA) in vitro. In accordance, TTD14 is localized in PI(3)P-enriched early endosomes in vivo, suggesting a role of this protein in vesicular sorting. Besides TTD14, Phospholipase D (PLD) and the retromer complex play a role in TRPL recycling. Loss of PLD activity results in enhanced degradation of TRPL. By contrast, in the retromer mutant TRPL transport to the ER seems to be compromised. Data obtained during the course of this project support previous findings regarding light- and rhodopsin-dependent degeneration in PLD3.1 mutant and in retromer-mutant photoreceptor cells. The mutation ttd14P75L causes light-dependent retinal degeneration as well. This degeneration of photoreceptors cells is associated with the TOR signaling pathway as was shown by genetic interaction studies. Interestingly, combination of reduced TOR activity with an as yet unidentified mutation X revealed a neuroprotective effect on ttd14P75L degeneration and rescued the TRPL trafficking defect of this mutant. In conclusion, the results of this work revealed a possible membrane protein recycling route via the ER and an unexpected contribution of the TOR signaling pathway to membrane protein trafficking and aspects of degeneration in neuronal cells.
Publications
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(2015) The GTP- and phospholipid-binding protein TTD14 regulates trafficking of the TRPL ion channel in Drosophila photoreceptor cells. PLoS Genet., 11(10): e1005578
Cerny, A.C., Altendorfer, A., Schopf, K., Baltner, K., Maag, M., Sehn, E., Wolfrum, U., and Huber, A.
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(2016). TTD14, a GTP- and phospholipid binding protein and the retromer complex are required for TRPL trafficking in Drosophila melanogaster photoreceptors. 2nd European Meeting on Phototransduction, Ascona (Schwitzerland), 04.- 07. September 2016
Schopf, K., Altendorfer, A., Baltner, K., Maag, N., Cerny, A.C., and Huber, A.
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(2017) Membrane protein trafficking in Drosophila photoreceptor cells. Eur. J. Cell Biol.
Schopf, K. and Huber, A.
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(2017). The Retromer complex, Phospholipase D and TTD14 Protein are required for endocytic recycling of TRPL in Drosophila melanogaster photoreceptors. H3 Symposium: Sensory transduction in insects, London (UK), 08. December 2017
Schopf, K., Smylla, T., and Huber, A.
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(2018). Retromer complex and Phospholipase D collaborate to recycle TRPL in Drosophila melanogaster photoreceptors, Neurofly, Krakau (Poland), 03.-07. September 2018
Schopf, K., Krieg, J., Smylla, T., and Huber, A.
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(2019) Immunocytochemical labeling of rhabdomeric proteins in Drosophila photoreceptor cells is compromised by a light-dependent technical artifact. J. Histochem. Cytochem.
Schopf, K., Smylla, T. K., and Huber, A.
