Abstract

The trafficking of proteins between vesicular compartments and delivery of proteins to particular subcellular locations are tightly regulated and essential for instance to achieve correct assembly of multimeric proteins and correct post-translational modifications. Arginine-based sorting motifs are shown to be critical in the biosynthetic processing of numerous transmembrane proteins. Schutze et al. first described a di-Arg ER-retention motif determined by two adjacent Arginines (RR) localized on one form of the invariant chain (Ii33, P04233) of the major histocompatibility complex (MHC) class II (Schutze, 8157008). The initial presumptions of a required proximity to the N terminus and specificity for type II membrane proteins have been superceded. The di-Arg motif is most commonly found on an N- or C-terminal cytosolic region but can be found at other cytosolic protrusions of polytopic membrane proteins (Michelsen, 16065065). In the context of studying assembly and trafficking of ATP sensitive K+ channels (K-ATP), Zerangue et al. found another variant of the di-Arg motif within the K-ATP subunits Kir6.1(Q15842), Kir6.2(Q14654) and SUR1(Q09428) (Zerangue, 10197533). Mutational analysis and chimeric fusion proteins revealed an RXR-based sequence retaining single K-ATP subunits and incorrectly assembled complexes in the ER, preventing surface expression.

The di-Arg motif is not only present in K-ATP channel subunits but also in subunits of additional heteromeric membrane proteins like GABA (Q9UBS5), Kainate (Q16478) and NMDA (Q05586) receptors. Here the di-Arg ER-localization motif functions as a quality control mechanism governing the ER exit. Single subunits of unassembled multimeric proteins with an exposed di-Arg motif, which is suggested to be recognized by generic eukaryotic trafficking machinery, are retained in the ER. Only properly folded and fully assembled protein complexes can leave the ER and reach the cell surface (Zerangue, 10197533; Taneja, 19357197). Therefore the di-Arg motif represents a checkpoint for both the coordination of sequential assembly of multimeric membrane proteins and the regulation of their delivery to the cell membrane defining the number of protein complexes at the cell surface (Scott, 11312291). Precise regulation mechanisms are still unknown, but a simple explanation for the regulation of di-Arg based ER-retention in terms of surface expression of multimeric proteins is an inhibition of the motif by steric masking during heteromerization. Furthermore, the interaction with additional proteins like coat protein complex I (COPI), 14-3-3 proteins as well as PDZ domain containing proteins may act as regulatory switches. For example, unassembled K-ATP subunits possessing an exposed RXR motif are shown to be recognized by the COPI machinery and retrieved to ER by COPI coated vesicles, defining the di-Arg motif as an ER retrieval and retention motif (Taneja, 19357197). By contrast, 14-3-3 proteins may be qualified to probe the assembly status of multimeric membrane proteins and are often required in order to negatively regulate the di-Arg motif. 14-3-3 proteins reduce COPI mediated retention, which is necessary for the forward transport of the assembled protein complexes (Yuan, 12699619; Taneja, 19357197, Heusser, 17038548). For some instances (ADAM22 (Q9P0K1) and GPR15 (P49685) the 14-3-3 binding sequence overlaps with the di-Arg ER-retention motif, again indicating a regulatory switch by steric masking controlled by basophilic kinases, since 14-3-3 proteins bind phosphopeptides. Additionally, the di-Arg motif present in an NR1 (Q05586) subunit can be masked by recruitment of PDZ domain containing proteins (Scott, 11312291). Finally, di-Arg based ER-retention is likely regulated by phosphorylation of nearby residues. Either phosphomimetic mutations or activation of PKC enhanced the surface delivery of NR1, suggesting an inhibitory effect on di-Arg based ER-retention (Scott, 11312291).

The CFTR protein is an unusual case. Cryptic di-Arg motifs are thought to become active due to cystic fibrosis unfolding mutations such as at F508 (10445036). The instance in ELM is annotated as a false positive as this di-Arg is not a normal cellular motif.