Dilysine Motifs Research Articles

SIGNAL-MEDIATED SORTING OF MEMBRANE PROTEINS BETWEEN THE ENDOPLASMIC RETICULUM AND THE GOLGI APPARATUS

Each organelle of the secretory pathway is required to selectively allow transit of newly synthesized secretory and plasma membrane proteins and also to maintain a unique set of resident proteins that define its structural and functional properties. In the case of the endoplasmic reticulum (ER), residency is achieved in two ways: (a) prevention of residents from entering newly forming transport vesicles and (b) retrieval of those residents that escape. The latter mechanism is directed by discrete retrieval motifs: Soluble proteins have a H/KDEL sequence at their carboxy-terminus; membrane proteins have a dibasic motif, either di-lysine or di-arginine, located close to the terminus of their cytoplasmic domain. Recently it was found that di-lysine motifs bind the complex of cytosolic coat proteins, COP I, and that this interaction functions in the retrieval of proteins from the Golgi to the ER. Also discussed are the potential roles this interaction may have in vesicular trafficking.

Delta- and zeta-COP, two coatomer subunits homologous to clathrin-associated proteins, are involved in ER retrieval.

Two new thermosensitive yeast mutants defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum (ER) were characterized. While both ret2-1 and ret3-1 were defective for ER retrieval, only ret2-1 exhibited a defect in forward ER-to-Golgi transport at the non-permissive temperature. Coatomer (COPI) from both mutants could efficiently bind dilysine motifs in vitro. The corresponding RET2 and RET3 genes were cloned by complementation and found of encode the delta and zeta subunits of coatomer respectively. Both proteins show significant homology to clathrin adaptor subunits. These results emphasize the role of coatomer in retrieval of dilysine-tagged proteins back to the ER, and the similarity between clathrin and coatomer coats.

Nonclathrin coat protein gamma, a subunit of coatomer, binds to the cytoplasmic dilysine motif of membrane proteins of the early secretory pathway.

Coatomer, a cytosolic heterooligomeric protein complex that consists of seven subunits [alpha-, beta-, beta'-, gamma-, delta-, epsilon-, and zeta-COP (nonclathrin coat protein)], has been shown to interact with dilysine motifs typically found in the cytoplasmic domains of various endoplasmic-reticulum-resident membrane proteins [Cosson, P. & Letourneur, F. (1994) Science 263, 1629-1631]. We have used a photo-cross-linking approach to identify the site of coatomer that is involved in binding to the dilysine motifs. An octapeptide corresponding to the C-terminal tail of Wbp1p, a component of the yeast N-oligosaccharyltransferase complex, has been synthesized with a photoreactive phenylalanine at position -5 and was radioactively labeled with [125I]iodine at a tyrosine residue introduced at the N terminus of the peptide. Photolysis of isolated coatomer in the presence of this peptide and immunoprecipitation of coatomer from photo-cross-linked cell lysates reveal that gamma-COP is the predominantly labeled protein. From these results, we conclude that coatomer is able to bind to the cytoplasmic dilysine motifs of membrane proteins of the early secretory pathway via its gamma-COP subunit, whose complete cDNA-derived amino acid sequence is also presented.

The Golgi-localization of yeast Emp47p depends on its di-lysine motif but is not affected by the ret1-1 mutation in alpha-COP.

The Saccharomyces cerevisiae EMP47 gene encodes a nonessential type-I transmembrane protein with sequence homology to a class of intracellular lectins defined by ERGIC-53 and VIP36. The 12-amino acid COOH-terminal cytoplasmic tail of Emp47p ends in the sequence KTKLL, which conforms with the consensus for di-lysine-based ER-localization signals. Despite the presence of this motif, Emp47p was shown to be a Golgi protein at steady-state. The di-lysine motif of Emp47p was functional when transplanted onto Ste2p, a plasma membrane protein, conferring ER localization. Nevertheless, the di-lysine motif was required for Golgi-localization of Emp47p and showed the same charge-independent, position-dependent characteristics of other di-lysine motifs. Alpha-COP has been shown to be required for ER localization of di-lysine-tagged proteins. Consistent with this finding, the Ste2p-Emp47p hybrid protein was mislocalized to the cell surface in the alpha-COP mutant, ret1-1. Surprisingly, the Golgi-localization of Emp47p was unaffected by the ret1-1 mutation. To investigate whether Emp47p undergoes retrograde transport from the Golgi to the ER like other di-lysine-tagged proteins we developed an assay to measure this step after block of forward transport in a sec12 mutant. Under these conditions retrograde transport led to a specific redistribution of Emp47p from the Golgi to the ER. This recycling occurred from a Golgi subcompartment containing alpha 1,3 mannose-modified oligosaccharides suggesting that it originated from a medial-or later Golgi compartment. Thus Emp47p cycles between the Golgi apparatus and the ER and requires a di-lysine motif for its alpha-COP-independent, steady state localization in the Golgi.

Targeting to the endoplasmic reticulum by dilysine motifs: role of coat proteins.

Targeting to the endoplasmic reticulum by dilysine motifs: role of coat proteins.

Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum

Dilysine motifs in cytoplasmic domains of transmembrane proteins are signals for their continuous retrieval from the Golgi back to the endoplasmic reticulum (ER). We describe a system to assess retrieval to the ER in yeast cells making use of a dilysine-tagged Ste2 protein. Whereas retrieval was unaffected in most sec mutants tested ( sec7, sec12, sec13, sec16, sec17, sec18, sec19, sec22, and sec23), a defect in retrieval was observed in previously characterized coatomer mutants ( sec21-1, sec27-1), as well as in newly isolated retrieval mutants ( sec21-2, ret1-1). RET1 was cloned by complementation and found to encode the α subunit of coatomer. While temperature-sensitive for growth, the newly isolated coatomer mutants exhibited a very modest defect in secretion at the nonpermissive temperature. Coatomer from β'-COP ( sec27-1) and α-COP ( ret1-1) mutants, but not from γ-COP ( sec21) mutants, had lost the ability to bind dilysine motifs in vitro. Together, these results suggest that coatomer plays an essential role in retrograde Golgi-to-ER transport and retrieval of dilysine-tagged proteins back to the ER.