Di‐lysine motif‐like sequences formed by deleting the C‐terminal domain of aquaporin‐4 prevent its trafficking to the plasma membrane

Abstract

Aquaporin‐4 is a transmembrane water channel protein, the C‐terminal domain of which is facing the cytosol. In the process of investigating the role of the C‐terminal domain of aquaporin‐4 with regard to intracellular trafficking, we observed that a derivative of aquaporin‐4, in which the C‐terminal 53 amino acids had been removed (Δ271‐323), was localized to intracellular compartments, including the endoplasmic reticulum, but was not expressed on the plasma membranes. This was determined by immunofluorescence staining and labeling of the cells with monoclonal antibody specifically recognizing the extracellular domain of aquaporin‐4, followed by confocal microscopy and flow cytometry. Deletion of additional amino acids in the C‐terminal domain of aquaporin‐4 led to its redistribution to the plasma membrane. This suggests that the effect of the 53‐amino acid deletion on the subcellular localization of aquaporin‐4 could be attributed to the formation of a signal at the C terminus that retained aquaporin‐4 in intracellular compartments, rather than the loss of a signal required for plasma membrane targeting. Substitution of the lysine at position 268 with alanine could rescue the Δ271‐323‐associated retention in the cytosol, suggesting that the C‐terminal sequence of the mutant served as a signal similar to a di‐lysine motif.

Structural and functional insights from the sequences and complex domain architecture of adhesin-like proteins from Methanobrevibacter smithii and Methanosphaera stadtmanae

“…The cytoplasmic di-Lysine motifs shown to be involved in tra cking of protein to ER and plasma membrane in earlier studies [23,24] was observed at the N-terminus of transmembrane helices in 22…”

A tyrosine‐based YXXΦ motif regulates the degradation of aquaporin‐4 via both lysosomal and proteasomal pathways and is functionally inhibited by a 10‐amino‐acid sequence within its C‐terminus

“…Moreover, replacing the C‐terminal domain with the corresponding region of mAQP1 (Fig. 1A) influences the steady‐state subcellular localization of AQP4 [23]. These observations suggest that the C‐terminal domain of AQP4 plays a role in its trafficking to lysosomes after antibodies bind against its extracellular domains.…”

“…We have demonstrated that deleting C‐terminal 53 amino acids also form a di‐lysine motif‐like sequence (Fig. 9A, Δ271–323) and prevents the mutant from localizing to the plasma membrane [23]. It should be noted that this mutant was quite stable and insensitive to protease inhibitors (Figs 2B,C (lane 5) and 7K,O), suggesting that a sequence implicated in destabilizing the protein exists between Gln 271 and Lys 313 .…”

“…1A, Lys 259 ‐Val 323 ) with the corresponding domain of mAQP1 (Fig. 1A, Phe 238 ‐Lys 269 ) influences the steady‐state subcellular localization of AQP4 [23]. These observations indicate an essential role for the C‐terminal domain in the subcellular localization and intracellular trafficking of AQP4.…”