Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer

Abstract

SummaryIn mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the À3,À4 position (relative to the cytosolic C-terminus) recruits in a very speci®c manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the À7,À8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.

What Is Moving in the Secretory Pathway of Plants?

“…This matches perfectly with earlier in vitro data (Contreras et al, 2004a(Contreras et al, , 2004b, but, interestingly, the previously identified diaromatic motif for in vitro COPII interaction was dispensable for ER export in vivo, despite the short length of the transmembrane domains (16 amino acids). ER export of membrane-spanning proteins is either very complex or current predictions of the transmembrane domain length is unreliable with available software.…”

The Golgi-Localized Arabidopsis Endomembrane Protein12 Contains Both Endoplasmic Reticulum Export and Golgi Retention Signals at Its C Terminus

“…Similarly, the localization of Emp46p family proteins to the Golgi in yeast was achieved via direct interaction of their dilysine signals with the COPI coat (Sato and Nakano, 2002). Indeed, the dilysine motifs (KKXX or KXKXX), which are usually located in the cytoplasmic C terminus of membrane proteins, have been shown to bind with the COPI subunit, especially the g-subunit of coatomer, in yeast, mammalian, and plant cells (Cosson and Letourneur, 1994;Harter and Wieland, 1998;Contreras et al, 2004a).…”

The Transmembrane Domain of the Molecular Chaperone Cosmc Directs Its Localization to the Endoplasmic Reticulum

“…ER retrieval systems include the C-terminal H/KDEL lumenal sequence (2,3), which is recognized by the ERD2-like receptor in post-ER compartments, leading to formation of COPIcoated vesicles and eventual retrotranslocation to the ER (4,5). Some ER-resident proteins carry other specific sequence motifs that interact with COPI complexes, such as di-lysine (K(X)KXX) (45,46), di-arginine (RR or RXR) (10,11), and diphenylalanine (FF) (13,14) that promote recruitment into COPI vesicle machinery for retrieval to the ER. Although relatively few type II resident membrane proteins, such as Cosmc, have been identified to date, some of these are known to be retained in the ER by the di-arginine motif located at the cytosolic N terminus, as seen for the MHC class II-associated invariant chain (10,11).…”

“…Both cytosolic di-arginine and lumenal determinants may be important for ER localization of some proteins such as Arabidopsis glucosidase I (12). Additional motifs include the di-phenylalanine (FF) motif (13,14), as seen in type I proteins like the p24 family, which are also associated with COPI coat protein binding.…”