An Analysis of Rhodopsin Trafficking in the Secretory Pathway

Rh1, the major rhodopsin in Drosophila, is the visual pigment expressed in R1-R6 photoreceptor cells. Rh1 is trafficked through the secretory pathway to its final destination in the rhabdomere. Visualization of the Rh1 trafficking process was examined in this work. In addition, the location and role of two proteins, Arf72, a small GTPase and the Drosophila tetracycline transporter known to affect Rh1 trafficking were probed. The initial effort involved developing a methodology to visualize rhodopsin during its progression through the secretory pathway. The visualization of Rh1 was accomplished by use of a heat shock promoter to drive transient expression of a Rh1:GFP tagged transgene (hs-Rh1-GFP). Intracellular membrane markers were also utilized to determine specific intracellular locations of rhodopsin. Co-expression of hs-Rh1:GFP and PDI:RFP (protein disulfide isomerase) confirmed localization to the endoplasmic reticulum at early time points. Co-localization of Rh1 and PDI was first visible at 2.5 hours post heat shock induction. After 3 hours, Rh1 began to progress beyond the ER before reaching the rhabdomere at approximately 10 hours. Rhabdomere localization was confirmed using labeled phalloidin to decorate the rhabdomere membranes. At 20 hours post heat shock induction, Rh1:GFP had completely localized to the rhabdomere. Thus, the earliest and final locations within the secretory pathway were labeled and co-localization was seen at the appropriate times. This data enabled us to approximate the time points when Rh1 would interact with cytoplasmic proteins suspected to play a role in rhodopsin trafficking.

An alternative approach was used to determine the intracellular location of Arf72. Arf72 is a member of the Ras superfamily of GTP binding proteins and required for rhodopsin transport. An Arf72:RFP transgene was created and co-expressed with characterized Golgi markers to identify specific location in the Golgi apparatus. Grasp65, a cis-Golgi marker, partially co-labeled with Arf72. In addition, Arf72 was expressed with Rab6, a medial/trans marker, but did not co-localize. To determine Arf72 function, Arf mosaic flies were generated. EM analysis of Arf72 mosaic flies show swelling of the ER and reduction in rhabdomere size. Co-expression of ER and Golgi markers were also expressed in Arf72 mutant photoreceptors. ER and Golgi distribution, as detected by the labeled markers described above, were unchanged in the Arf mutant background. A series of experiments were also performed on the tetracycline transporter (Tet). Tet was determined to play a role in Rh1 maturation as the gene disruption in Tet resulted in an overall reduction in Rh1 production. Electron microscopy also revealed changes in rhabdomere morphology. Co-expression of intracellular markers resulted in uncharacteristic Rab6 expression and localization. These results extend the characterization of the rhodopsin maturation process and provide additional insights to the roles of Arf72 and Tet proteins.