EMC is required for biogenesis of Xport‐A, an essential chaperone of Rhodopsin‐1 and the TRP channel

Abstract

The ER membrane protein complex (EMC) is required for the biogenesis of a subset of tail anchored (TA) and polytopic membrane proteins, including Rhodopsin-1 (Rh1) and the TRP channel. To understand the physiological implications of EMCdependent membrane protein biogenesis, we perform a bioinformatic identification of Drosophila TA proteins. From 254 predicted TA proteins, screening in larval eye discs identified two proteins that require EMC for their biogenesis: fan and Xport-A. Fan is required for male fertility in Drosophila and we show that EMC is also required for this process. Xport-A is essential for the biogenesis of both Rh1 and TRP, raising the possibility that disruption of Rh1 and TRP biogenesis in EMC mutants is secondary to the Xport-A defect. We show that EMC is required for Xport-A TMD membrane insertion and that EMC-independent Xport-A mutants rescue Rh1 and TRP biogenesis in EMC mutants. Finally, our work also reveals a role for Xport-A in a glycosylation-dependent triage mechanism during Rh1 biogenesis in the endoplasmic reticulum.

Xport-A functions as a chaperone by stabilizing the first 5 transmembrane domains of Rhodopsin-1

“…Based on the results of (Gaspar et al , 2022) and the results described here we favor a model where Xport-A acts as chaperone during the biogenesis of Rh1, at a transient step, when the TMDs 1-5 of Rh1 are already inserted in the ER membrane, but TMDs 6-7 are not yet inserted or at least not yet present in Rh1 structure. Interactions of Xport-A with Rh1 TMD1-5 must be essential to stabilize the TMDs of Rh1 but also to stabilize the N terminal ER luminal domain of Rh1 and the beta-loop-beta motif between TMD4 and TMD5, allowing for the correct folding and biogenesis of Rh1.…”

“…4A) are a series of mutants which lead to an increasingly more hydrophobic TMD of Xport-A, progressively bypassing the EMC requirement for membrane insertion. We have also shown that Xport-A 2L and 4L rescue the expression of Rh1 in EMC mutant cells (Gaspar et al , 2022), but in both cases these rescues were only partial, suggesting that these 2L and 4L mutants have reduced function, although they are inserted into the membrane, even in EMC mutant cells (Gaspar et al , 2022). In Fig.…”

“…Recently we have shown that the ER membrane protein complex (EMC) is required for the biogenesis and membrane insertion of Xport-A (Gaspar et al , 2022). In the last figure of that manuscript, we expressed 3 truncations of Rh1 (TMD1, TMD1-3 and TMD1-5) (Hiramatsu et al , 2019) in the background of flies heterozygous or homozygous for XportA 1 mutation.…”

“…Based on our findings in (Gaspar et al , 2022), we hypothesized whether Xport-A could be required for Rh1 biogenesis at the stage when the first 5 TMDs of Rh1 are inserted into ER membrane, rather than later, when all TMDs of the full length (FL) Rh1 are inserted into the membrane. To provide insights into this issue we resorted to AlphaFold2 (Jumper et al , 2021; Varadi et al , 2022) structural predictions of the Xport-A TMD together with full length (FL) Rh1 or Rh1 TMD1-5 (Fig.…”

“…2B. Of particular interest, are the positions of the amino acid residues that we previously mutated to Leucine (Gaspar et al , 2022). Xport-A 1L, 2L, 3L and 4L (Fig.…”