Most coronin proteins rely on interaction with actin in their functions. Mammalian coronin 7 has not been shown to interact with actin, but rather to bind to the outer side of Golgi complex membranes. Targeting of coronin 7 to Golgi membranes requires the activity of Src kinase and integrity of AP-1 adaptor protein complex. Coronin 7 further physically interacts with both AP-1 and Src in vivo and in vitro and is phosphorylated by Src. Depletion of coronin 7 by RNAi results in Golgi breakdown and accumulation of arrested cargo proteins, suggesting the protein functions in the later stages of cargo sorting and export from the Golgi complex. We suggest that coronin 7 acts as a mediator of cargo vesicle formation at the trans-Golgi network (TGN) downstream of AP-1 interaction with cargo but upstream of protein kinase D dependent membrane fission.

Introduction

The ability to bind actin is an intrinsic capacity of coronin proteins (reviewed in refs. 1, 2). In some family members, it is often hard to define the bona fide actin binding domain because most parts of the molecule possess actin binding properties.^3-5 There is at least one family member, however, which until to date has not been shown to physically interact or colocalize with actin. Although it is quite possible that future research will reveal specific conditions, processes or cell types where mammalian coronin 7 (CRN7; current official symbol: CORO7) associates with actin cytoskeleton, the current data suggest that this family member is unique in that its function is irrelevant to the regulation of the cytoskeleton.

Molecular Architecture

Coronin 7 belongs to the subgroup of “longer”, i.e., double-core coronins.¹ The human protein consists of 925 amino acids and displays a molecular architecture similar to that of Drosophila melanogaster and Caenorhabditis elegans POD-1 proteins (Fig. 1A). It is 46% and 47% homologous and 30% and 29% identical to Drosophila Dpod-1 and C. elegans POD-1, respectively. The regions of highest homology to the predicted seven-blade propeller structure⁶ of murine coronin 1 (coronin 1A, p57) correspond to amino acids 7-344 and 471-815 in human coronin 7, implying a double seven-blade propeller. The unique feature of the human coronin 7 is a 47 amino acid long low complexity proline-, serine- and threonine-enriched region in the intermediate region of the molecule (amino acids 425-472), N-terminal to the second propeller. Like both POD-1 coronins, mammalian coronin 7 lacks the C-terminal coiled-coil domain. There are no predicted transmembrane domains or signal peptide sequences.

Figure 1

Molecular architecture and phylogenetic analysis of coronin 7 proteins. A), Domain structure of human coronin 7. Core domains consisting of WD repeats are preceded by “coronin signature” motifs. Note the low complexity proline, serine- (more...)

Software predictions^7-9 suggest a number of potential phosphorylation sites in the coronin 7 protein. The highest E value predictions are for potential MAP kinase phosphorylation sites at serine residue 442 and threonines 497 and 733, cdc2 sites at S-450 and S-775, cdk5 at S-437, PKC sites at S-7, S-465 and T-654 and Src sites at tyrosine residues 288 and 758. Not surprisingly, many serine and threonine phosphorylation predictions concentrate in the low complexity PST-enriched region. Although it remains to be elucidated which of the predicted sites are relevant in vivo, at least some of them have been experimentally demonstrated to be phosphorylated in vitro (see below).

Tissue Distribution and Subcellular Localization

Coronin 7 has been detected in most murine tissues by western blot, with the notable exception of heart and skeletal muscle, and the corresponding mRNA was found to be strongly enriched in the brain, kidney and thymus.¹⁰ In cultured mammalian cells, the protein is easily detected by immunofluorescence in the Golgi/TGN area partially colocalizing with both Golgi and TGN markers and this localization is abolished in cells treated with brefeldin A, a fungal metabolite known to fuse Golgi membranes with endoplasmic reticulum.^10,11 Additional analyses revealed that coronin 7 resides at the outer side of Golgi membranes where it could be detected by immunoelectron microscopy (VR, unpublished observations). Additionally, the protein is not protected from proteolytic cleavage by the compartment membrane (Rybakin et al, submitted for publication).

A substantial amount of coronin 7 is additionally present in unidentified cytoplasmic “spots” probably corresponding to large protein complexes or very small vesicles of unknown nature. Subcellular fractionation experiments showed that the bulk of the protein is actually cytosolic and only a minor fraction coincides with a detergent-soluble membrane pellet. Interestingly, in contrast to the cytosolic pool, membrane-associated coronin 7 was found to be phosphorylated on tyrosine residues, as demonstrated by two-dimensional gel electrophoresis and western blot with anti-phosphotyrosine antibodies.¹⁰ This finding led to the conclusion that tyrosine phosphorylation may either be required for membrane targeting of coronin 7 or for its function on membranes. As discussed above, the strongest predicted tyrosine phosphorylation sites were found at tyrosine residues 288 and 758.

In vitro phosphorylation experiments have been performed in order to find out whether predicted phosphorylation sites are biologically relevant. Because tyrosine phosphorylation has been shown to coincide with membrane localization of coronin 7, various tyrosine kinase activities were tested for their ability to influence coronin 7 targeting. Firstly, it has been established that nonspecific inhibition of tyrosine protein kinases resulted in the decrease in membrane-bound coronin 7. Additionally, SU6656, a specific inhibitor of Src family kinases showed the same effect; moreover, it has been demonstrated that expression of dominant negative Src resulted in the redistribution of coronin 7 to the cytosol (Rybakin et al, submitted for publication). These data suggested that Src kinase activity may be required to bring coronin 7 to membranes. Further experiments showed that purified recombinant Src was able to specifically phosphorylate tyrosine 288 as well as 758 of synthetic coronin 7 peptides and phosphorylation of tyrosine 758 was significantly stronger than that of tyrosine 288. Full-length recombinant coronin 7 also is phosphorylated by purified Src in vitro. Additionally, endogenous Src and coronin 7 could be co-immunoprecipitated from HeLa cells (Rybakin et al, submitted for publication). Together, these data suggest that phosphorylation of coronin 7 by Src may be a key mechanism regulating the recruitment of coronin 7 from the cytosol to Golgi membranes.

The Function of Coronin 7 in the Biosynthetic Pathway

The Golgi complex is the central protein sorting organelle in mammalian cells. Total protein input from the endoplasmic reticulum reaches the cis-side of the compartment and cargo proteins gradually traverse the Golgi while being sequentially modified. It is believed that protein modification, most importantly glycosylation, provides cargo proteins with “sorting signals” which are recognized by luminal binding sites of cargo receptors and define the Golgi export route that the cargo will take. Additional sorting clues are provided by short amino acid motifs exposed to the cytosolic side of the Golgi membrane. Upon reaching the trans-Golgi network, cargo proteins are sorted into distinct subpopulations of transport intermediates according to their sorting signals.

The Golgi localization of coronin 7 and its interaction with the sorting machinery suggested that the protein could participate in the regulation of protein trafficking along the biosynthetic pathway. RNAi experiments demonstrated that transient depletion of coronin 7 is sufficient for an effective inhibition of protein export from the Golgi. An anterograde trafficking marker, vesicular stomatitis virus glycoprotein G (VSVG), has been shown to accumulate in the Golgi/TGN zone and its further transport is severely affected. Quantification of VSVG fluorescence at the cell surface showed that indeed most of the protein does not reach plasma membrane upon coronin 7 RNAi. Apart from protein trafficking defects, cells treated with coronin 7 siRNA exhibit marked defects in Golgi morphology.¹¹ As shown by immunofluorescence and electron microscopy, perinuclear Golgi ribbons are disintegrated and short mini-stacks positive for both cis- and trans-Golgi markers are scattered throughout the cytosol. A similar morphology has been previously described in cells treated with the microtubule depolymerizing agent nocodazole. However, in nocodazole treated cells cargo progression is not perturbed.¹² It is highly unlikely that trafficking defects in coronin 7 RNAi cells are due to ineffective or incomplete glycosylation. In RNAi experiments, the marker cargo protein LAMP1^13,14 has been shown to be properly glycosylated in the absence of coronin 7, while a knockdown of a COG3 protein known to participate in the glycosylation in the Golgi¹⁵ resulted in the decrease of LAMP1 glycosylation.¹¹

Because of the lack of a signal peptide or hydrophobic regions, coronin 7 protein is likely to be linked to the outer side of the compartment membrane by means of interaction with another membrane protein or proteins. This assumption has been experimentally confirmed by the finding that coronin 7 interacts with the AP-1 adaptor protein complex in vivo and in vitro.¹¹ The heterotetrameric AP-1 complex associates with cytosolic tails of biosynthetic cargo and cargo receptors en route from the TGN to late endosomes and lysosomes. μ-subunits of AP complexes are known to specifically bind to YxxΦ motifs present in cytosolic tails of AP-1 cargo molecules.¹⁶ Interestingly, coronin 7 possesses two YxxΦ motifs located in the C-terminal parts of each WD repeat propeller (288-291, 758-761) and is therefore theoretically capable of interacting with AP-1 via its μ1 subunit. Indeed, coronin 7 was detected in complex with AP-1 precipitated using an antibody recognizing γ 1-adaptin and, reversely, γ 1-adaptin was detected coprecipitated together with coronin 7. Additionally, direct and specific interaction between peptides harboring coronin 7 YxxΦ motifs and purified μ1 has been demonstrated in surface plasmon resonance experiments. The predicted sorting motif based on tyrosine 288 showed the strongest binding to the AP-1μ subunit.¹¹

The interaction with the AP-1 complex appears biologically significant because in coronin 7 RNAi cells, the AP-1 dependent cargo molecule mannose-6-phosphate receptor MPR46 is accumulated in the Golgi zone and this accumulation coincides with increased AP-1 staining at the TGN.¹¹ Depletion of μ1-adaptin resulted in the dispersal of coronin 7 from Golgi membranes (Rybakin et al, submitted for publication) implying that the integrity of AP-1 and its membrane localization is indeed required for the Golgi targeting of coronin 7. It is reasonable to believe that coronin 7 functions immediately downstream of the cargo binding to the AP-1 complex, allowing the transport intermediate to form and/or detach.

Coronin 7 Mediated Regulation of Golgi Function: Species-Specific Mechanisms?

It has been convincingly demonstrated that the mammalian coronin 7 is localized to the Golgi complex and regulates protein traffic in the anterograde direction.^10,11 Chapter 7 summarizes the functions of invertebrate coronin 7 orthologs. Indeed, there is evidence that nonmammalian orthologs may function in the biosynthetic pathway as well. This is best illustrated by the C. elegans phenotype where POD-1 (coronin 7) deletion results in defects in the formation of the embryonic eggshell and accumulation of vesicular structures within the egg. It has been shown that in mammalian cells, two critical residues are essential for the interaction of coronin 7 with the AP-1 complex and Src protein kinase. Tyrosine 288-basedsignal appears to be the key motif for binding of coronin 7 to the µ subunit of AP-1,¹¹ while tyrosine 758 is the crucial Src phosphorylation site (Rybakin et al, submitted for publication). Are these residues conserved?

Phylogenetic analysis (Fig. 1B) demonstrates that the conservation of the amino acid sequences in the vicinity of tyrosines 288 and 758 among invertebrates is very low. Although the Drosophila sequence features a slightly shifted YxxΦ motif not identical to that of human coronin 7, there is no YxxΦ motif at the vicinity of the residue 288 in the C. elegans POD-1 sequence. Moreover, the Src target tyrosine Y758 in the human sequence is conserved in Drosophila but absent in C. elegans. This suggests that the coronin 7 ortholog in Drosophila may be similar to the human protein as far as its regulation is concerned, but the worm ortholog appears to function in a different manner and/or to be completely differently regulated.

Interestingly, although coronin 7 proteins are very well conserved among mammalian species, both tyrosine residues implicated in the regulation of coronin 7 are located in higher variability regions (Fig. 1C). Although the tyrosine 758 is well conserved as well as its vicinity, the tyrosine-based YxxΦ motif at tyrosine 288 is not present in the mouse and rat protein sequence because of the change in the fourth (Φ) amino acid. Interestingly, the YxxΦ motif based on tyrosine 758 shown to bind to µ1 albeit with a lower affinity than tyrosine 288¹¹ is well conserved in most mammalian species including mouse and rat and may indeed participate in the binding to the adaptor complex. It will be interesting to decipher the relationship between the phosphorylation by Src and binding to AP-1 in the case of tyrosine 758. It may well be possible that phosphorylation will dramatically increase the affinity of AP-1 to the conserved tyrosine 758-based sorting signal.

Future Directions

The exact mechanism that is involved in the coronin 7 mediated regulation of cargo exit from the Golgi complex remains to be elucidated. One of several possible mechanisms includes a role in the formation of a clathrin cage around the AP-1 complexed with cargo. Future experiments will need to address the question whether there is a clathrin coat accumulating in Golgi remnants in coincidence with arrested cargo and AP-1. Additionally, it has been suggested that coronin 7 may be a part of the protein kinase D related machinery regulating the formation of basolateral transport intermediates at the TGN.^17,18 It is unclear how the AP-1/clathrin system interacts with PKD-dependent mechanisms of transport carrier formation and it is not even clear whether the two ever participate in the regulation of the formation of the same vesicle. It has, however, been established that at least basolateral protein transport in polarized cells requires both protein kinase D activity¹⁸ and adaptor protein complexes.^19,20 Coronin 7 may constitute the missing link between the AP complex binding to the cargo at the TGN and activation of diacylglycerol-dependent recruitment and subsequent activation of PKD resulting in the membrane fission.

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