Golgi Export Signal Ties Proper Folding to AP1 Binding
Golgi Export of the Kir2.1 Channel Is Driven by a Trafficking
Signal Located Within Its Tertiary Structure
Ma et al
A newly defined Golgi export signal uncovers an important quality control mechanism and identifies a novel AP1 interaction.
The trafficking of ion channels to and from the cell surface has been an active area of investigation for some time now. A great deal has been accomplished in mapping some of the routes followed by various channels to and from the cell surface, in identifying motors and regulators with which they interact, and in defining trafficking signals within the channels themselves.1–3 Among the trafficking signals harbored within ion channels themselves, signals for retention in and trafficking out of the endoplasmic reticulum (ER) are the best understood. Both types consist of short, linear amino acid sequences and appear to be relatively simple in their operation. ER retention signals, for example, are thought to be generally exposed only in misfolded or not fully assembled ion channel complexes,4 and we have substantial mechanistic insights into how several of these signals operate. In addition to interfering with ER exit itself, RXR ER-retention signals, for example, have been shown function through differential affinities for 14-3-3 and COPI proteins; 14-3-3 proteins exhibit high affinities for RXR motifs in properly assembled channels and promote delivery of the channels to the Golgi. Conversely, COPI appears to exhibit high affinity for improperly folded channels and promotes the return of the minority of the channels that escape the ER back to that organelle.5,6 The ER exit signal, Aspartate-Alanine-Aspartate, of the CFTR channel is required for interaction with Sec23/24, and is thus thought to promote the COPII-dependent trafficking of that channel to the Golgi apparatus. Our understanding of the mechanisms regulating the trafficking of channels once they reach the Golgi is much less well developed.
Although several mutations are known to cause channels to be retained in the Golgi apparatus,7–10 an understanding of how they lead to this phenotype has been lacking. A short sequence in the Kir2.1 inward rectifier potassium channel has been identified as required for Golgi export,7 but the mechanism by which it acts has been a mystery until now. Involving especially residues R44 and R46 in the channel's N-terminal cytoplasmic domain, deletions and specific mutations of this sequence cause retention in the Golgi, despite the fact that the channels appear to fold and assemble normally.7 In an extremely thorough series of experiments, Ma et al11 have now elegantly determined how this sequence operates. Functioning in conjunction with a group of amino acid residues much distal to it in Kir2.1, this domain is involved in the recruitment of the AP1 clathrin adaptor and thereby promotes the exit of properly folded channels from the Golgi apparatus.
Ma et al11 gained their primary insight in examining the crystal structure of the Kir2.1 cytoplasmic domains.12 They noticed that the N-terminal motif identified by Stockklausner and Klocker7 was in close juxtaposition in the folded structure to residues 314 and 315 of the C-terminal domain of the channel. This was of particular interest because deletion of residues 314 and 315 has been shown to cause Kir2.1 channels to be retained in the cytoplasm,13 although the locale of that retention had not been determined. The deletion was first identified in patients with Andersen-Tawil syndrome.14
Hypothesizing that these regions in the N- and C-terminus may function together, Ma et al11 first tested whether the Δ314–315 mutation, like mutations in R44 and R46, causes Kir2.1 channels to accumulate in the Golgi. Indeed, using a combination of immunocytochemical, live cell imaging, pulse-chase, and glycosylation assays, Ma at al, clearly establish that the mutant channels accumulate in the medial and trans-Golgi. The blockage, they determined, is in the transport of the mutant channels from the trans-Golgi to the terminal cisternae. The phenotype of the Δ314–315 mutation is thus very similar to that of the previously reported mutations in R44 and R46.
Ma et al11 went on to establish that the presence of both cytoplasmic segments is required for successful Golgi export. When the Kir2.1 N- and C-terminal domains were grafted individually or as a combined fusion construct onto the human C-lymphocyte membrane protein CD8, neither the CD8-Kir2.1 N-terminal construct nor the CD8-Kir2.1 C-terminal construct trafficked to the cell surface. Both instead colocalized with a Golgi marker. CD8 fused to the combined Kir2.1 N- and C-terminal domains, in contrast, exhibited robust surface expression. Thus, the N- and C-terminal domains appear able to promote Golgi export only if they are present together. To more directly test the hypothesis that the juxtaposed N- and C-terminal residues themselves are required for this export, Ma et al11 assayed the effects of individual mutations at positions in the immediate vicinity of residues 314 and 315 as informed by the channel's crystal structure.12 They found that mutations in 6 of these residues resulted in accumulation of the channels in the Golgi apparatus. Most of these residues reside on the surface of the folded protein, where they form a contiguous patch, involving charged residues at one end and a shallow, hydrophobic cleft at the other. A potential recognition site for an interacting protein, comprised of residues from 2 disparate regions in the Kir2.1 primary sequence, has been identified that very probably forms only in properly folded channels.
A crucial question, of course, is which potentially interacting protein(s), if any, bind to this patch. Ma et al11 tested whether AP1 or GGA1, well established to be involved in cargo export from the trans-Golgi,16 were able to bind to a Kir2.1 N-,C-terminal domain fusion construct. AP1, they found, did indeed bind to the construct but GGA1 did not bind to the construct. AP1 did not bind, however, to an otherwise equivalent construct harboring the Δ314–315 deletion nor to constructs harboring mutations in other residues of the surface patch. This is strong circumstantial evidence that the wild-type patch is indeed an AP1 binding site. Further experiments identified the γσ1 to be the subunit of AP1 that binds to the combined N-,C-terminal domains of Kir2.1. No binding of the Kir2.1 domains to the β1μ1 subunit was detectable.
Confirming that AP1 is indeed required for Kir2.1 trafficking, Ma et al demonstrated that RNA-knockdown of AP1-γ-adaptin reduced surface expression of wild-type Kir2.1 and caused an accumulation of the channel at the trans-Golgi. Temperature block/release experiments demonstrated that AP1 and the wild-type channel interact only when they become competent to exit the Golgi apparatus.
It is highly likely that formation of the patch identified by Ma et al is key for the recognition of properly folded Kir2.1 and for its selective export from the Golgi apparatus. Because AP1 recognizes the patch through a novel interaction with its γσ1 subunit, Ma et al11 argue that it is free also to interact with other cargo at the same time, through interactions of its μ1 subunit with canonical “YXXφ” signals, well established as AP1 binding sites in other proteins.16 The trafficking process thus could be more efficient and perhaps subject to novel controls. This is a very interesting suggestion but remains highly speculative.
A strong alternative possibility is that the novel surface patch may instead act in concert with conventional AP1 binding sites present in the channel. Each subunit of Kir2.1 harbors a YXXφ signal, YIPL, at positions 242–245 in the C-terminal cytoplasmic domain. Mutations in this signal have been shown to dramatically reduce Golgi export of Kir2.1.9 This YXXφ signal in Kir2.1 is, however, not entirely typical. Unlike the great majority of such signals,16 neither of the “X” residues within it is polar. Thus, it is quite possible that the affinity of the AP1 μ1 subunit for the signal is weak. Perhaps the novel interaction of the patch with the AP1 γσ1 subunit acts to increase the overall affinity of AP1 for the properly folded channel. It will be important to test whether the patch can promote Golgi export in the absence of the YIPL signal.
Ma et al also speculate that they may have found a new function for AP1. Because in the COS7 cells, Kir2.1 traveled rapidly to the plasma membrane on exit from the Golgi and did not appear to accumulate in endosomes, Ma et al11 argue that AP1 may be involved in direct transport from the trans-Golgi to the cell surface, independent of endosomes. This would indeed be a new function for the clathrin adaptor, but their evidence is weak. It remains possible that the authors merely failed to detect a very rapid route through endosomal compartments. No attempt was made to identify colocalizations with endosomal markers. Thus, evaluation of the possibility of a direct route to the cell surface will have to await further experimentation.
In summary, Ma et al have identified a novel mechanism by which export from the Golgi apparatus is tied to the correct folding of a membrane protein. Whereas, as Ma et al point out, conformational signaling in SNARE proteins is common in vesicle sorting,17–19 this is the first example of Golgi-export regulation by such a mechanism. It will be of great interest to learn whether similarly constituted signals are widely used in regulating the export of other ion channels and membrane proteins from the Golgi. The mechanism is likely to be operative in other Kir channels; the involved residues are mostly conserved across members of the Kir1, 2, 3, and 6 families.15 Intriguingly, in Kir2.4, which is trafficked less efficiently than Kir2.1, the residue at the position equivalent to R46 in Kir2.1 is a valine.9
The finding that Golgi export can depend on the formation of a recognition signal from disparate regions in a protein's primary structure is a significant advance in our understanding of intracellular quality control mechanisms. We look forward to future work elucidating the mechanism by which AP1 recognizes the novel patch that Ma et al have identified and of the route through which it catalyzes Kir2.1 trafficking to the cell surface.
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Commentaries serve as a forum in which experts highlight and discuss articles (published here and elsewhere) that the editors of Circulation Research feel are of particular significance to cardiovascular medicine.
Commentaries are edited by Aruni Bhatnagar and Ali J. Marian.
- © 2012 American Heart Association, Inc.
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- Mohapatra DP,
- Trimmer JS
- Stockklausner C,
- Klocker N
- Cheng J,
- Moyer BD,
- Milewski M,
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- Cutting GR,
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- Stanton BA,
- Guggino WB
- Hofherr A,
- Fakler B,
- Klocker N
- Bendahhou S,
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- Plaster NM,
- Tristani-Firouzi M,
- Fu YH,
- Ptacek LJ