Multiple plasma membrane reporters discern LHFPL5 region that blocks trafficking to the plasma membrane

Abstract

The mechano-electrical transduction (MET) channel of the inner ear receptor cells, termed hair cells, is a protein complex that enables our senses of hearing and balance. Hair cell MET requires an elaborate interplay of multiple proteins that form the MET channel. One of the MET complex components is the transmembrane protein LHFPL5, which is required for hair cell MET and hearing. LHFPL5 is thought to form a multi-protein complex with other MET channel proteins, such as PCDH15, TMIE, and TMC1. Despite localizing to the plasma membrane of stereocilia, the mechanosensing organelles of hair cells, LHFPL5 requires its binding partner within the MET complex, PCDH15, to localize to the stereocilia tips in hair cells and to the plasma membrane in heterologous cells. Using the Aquaporin 3-tGFP reporter (AGR) for plasma membrane localization, we found that a region within extracellular loop 1, which interacts with PCDH15, precludes the trafficking of AGR reporter to the plasma membrane in heterologous cell lines. Our results suggest that the presence of protein partners may mask endoplasmic reticulum retention regions or enable the proper folding and trafficking of the MET complex components, to facilitate expression of the MET complex at the stereocilia membrane.

Figure 1

AGR discerns the ability of LHFPL5 regions to halt trafficking to the PM. AGR-based method was used to examine whether any of intracellular and extracellular loops, as well as N- and C-termini of LHFPL5 have the ability to preclude any detectable reporter trafficking to the PM. (A) The N-terminus fragment of LHFPL5, comprising residues LHFPL5^1–23, does not preclude trafficking of the AGR system to the PM. When expressed in HEK293 cells, AQP3-tGFP-LHFPL5^1–23 exhibit robust PM localization (white arrows). (B) In contrast, extracellular loop 1, consisting of LHFPL5^47–96 fragment, halts any detectable trafficking to the PM. AQP3-tGFP-LHFPL5^1–23 expression shows an intracellular only labeling pattern. (C–E) AGR-based constructs containing the remaining intracellular loop 1, LHFPL5^120–125 (C), extracellular loop 2, LHFPL5^149–176 (D) or the C-terminus, LHFPL5^200–219 (E) show PM labeling. Scale bars: 10 µm. ER was labeled with ER-red fluorescent protein (ER-RFP) in all figures.

Figure 2

Employing E-Cadherin-mGFP- and mGFP-CD10-based reporters to test specific protein regions. (A) E-Cadherin belongs to the Type-I transmembrane family of proteins. E-Cadherin-mGFP (monomeric GFP) displays strong PM localization in transiently transfected HEK293 cells (white arrows), similar to the AGR system. E-Cadherin-mGFP reporter could be used to re-test intracellular loop domains and termini. (B) CD10 belongs to the family of Type-II transmembrane proteins. mGFP-CD10 shows intense PM localization in HEK293 cells. mGFP-CD10 reporter could be used to test extracellular loop domains and termini. Scale bars: 8 µm.

Figure 3

E-Cadherin- and CD10-based reporters confirm intracellular retention quality of the extracellular loop 1, LHFPL5^47–96. (A) N-terminus of LHFPL5, residues LHFPL5^1–23, when fused to the C-terminus of E-Cadherin-mGFP does not preclude reporter PM localization, confirming result obtained with AGR. (B) When extracellular loop 1 fragment LHFPL5^47–96 was fused to C-terminus of mGFP-CD10 reporter, no detectable PM labeling was observed. These results confirm the data acquired using AGR, corroborating the ability of the LHFPL5^47–96 to halt trafficking to the PM. (C–E) Intracellular loop 1 fragment LHFPL5^120–125 (C), extracellular loop 2 fragment LHFPL5^149–176 (D), and C-terminus fragment LHFPL5^200–219 (E) did not prevent corresponding reporters E-Cadherin-mGFP-LHFPL5^120–125 (C), mGFP-CD10-LHFPL5^149–176 (D), and E-Cadherin-mGFP-LHFPL520^200–219 (E) trafficking to the PM, confirming the result obtained by AGR. Scale bars: 10 µm.

Figure 4

Further scanning ECL1 region LHFPL5^47–96 to identify the exact fragment that prevents trafficking to the PM. (A) ECL1 fragment LHFPL5^47–62 does not prevent reporter trafficking to the PM. When HEK293 cells were transfected with AQP3-tGFP-LHFPL5^47–62, robust PM localization was observed (arrows). (B–D) Likewise, ECL1 fragments LHFPL5^59–74 (B), LHFPL5^71–86 (C), and (D) LHFPL5^83–96 did not prevent PM localization (arrows). Scale bars: 8 µm.

Figure 5

Assessing Transmembrane Segments of LHFPL5 for the ability to halt trafficking to the PM using AGR. (A) The first TM segment comprising residues of LHFPL5^16–45 does not prevent reporter trafficking to the PM. PM localization was detected in HEK 293 cells transfected with AQP3-tGFP-LHFPL5^16–45 (arrows). (B–D) Second TM segment amino acid sequence LHFPL5^95–121 (B), third TM sequence LHFPL5^126–154 (C) and fourth TM sequence LHFPL5^180–201 (D) also did not prevent PM localization. Scale bars: 10 µm.