Abstract
Purpose :
Connexin36 (Cx36) containing gap junctions are critical components for retinal signal processing, providing unique functions such as signal averaging or cell synchronization. While the physiological features of gap junctions in electrically coupled networks have been intensively studied, little is known about the molecular mechanisms that control the transport of connexins in retinal neurons. Previous studies have shown that expression of a Cx36-EGFP construct carrying a dysfunctional C-terminus interferes with the formation of electrical synapses in the retina. These observations indicate that a fully functional C-terminus in Cx36 is necessary to deliver the connexin to the synapse. The underlying molecular pathway responsible for the transport deficit is currently unknown and subject of the present study.
Methods :
Site directed mutagenesis and HEK293T cells serving as an expression system were used to characterize transport deficits of individual Cx36 mutants. A proximity labeling strategy in combination with mass spectrometry was used to identify novel proteins that associate with the carboxy terminus of Cx36. Functional interactions with candidate proteins were verified by siRNA screens and pull-down assays. For each experimental condition, confocal scans were acquired to analyze the distribution of Cx36 in transfected HEK293 cells.
Results :
Expression of Cx36 mutants that lacked tyrosine and valine at the extreme C-terminus led to an intracellular accumulation in transfected HEK293T cells. Double labeling experiments demonstrated that these aggregates originated from the ER. To search for proteins that mediate the transport of Cx36 at ER exit sites we performed a BioID assay using a Cx36-TurboID construct. This screen identified several COPII components, including cargo receptors and the Golgi stacking protein GRASP55. siRNA mediated knockdown of all four COPII cargo receptors, revealed that only the combined depletion of Sec24A and B blocked the ER export of Cx36.
Conclusions :
In the present study we have dissected the molecular mechanism that guides the transport of Cx36 in the early secretory pathway. Our data indicate that a C-terminal motif, comprising the amino acids tyrosine and valine, is required for Cx36 to bind specific cargo receptors of the COPII complex enabling the recruitment of newly synthesized connexons into ER export vesicles.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.