Candidate AP-5 heterotetramer appears to be involved in late endosomal trafficking and is implicated in human disease.
Researchers have identified what they claim is a fifth adaptor protein complex involved in shuttling cellular cargo from one organelle to another. They say that the complex, which they’ve designated AP-5, localizes to a late endosomal compartment, and its subunits can be found in all five eukaryotic supergroups.
The team was composed of scientists at the University of Cambridge and the University of Alberta. Reporting in PLoS Biology, Joel Dacks, Ph.D., along with Margaret Robinson, Ph.D., and colleagues suggest that AP-5 is an evolutionarily ancient complex, which has nevertheless been lost in many organisms.
“What this does for cell biology is open up a whole new avenue of research,” Dr. Dacks claims. “We thought there were four big players in the processes of how things got moved around in the back half of the cell. There’s a fifth player on the field; we just couldn’t see it.” In their paper, titled “The Fifth Adaptor Protein Complex,” the researchers in addition report on hereditary spastic paraplegia, in which AP-5 appears to play a role.
Adaptor protein (AP) complexes are responsible for sorting cellular cargo into vesicles for transport from one membrane compartment to another, including the trans-Golgi network (TGN), the endosome, the lysosome and the plasma membrane, the researchers explain. Four distinct heterotetramer AP complexes (AP-1 through to AP-4) have been identified in eukaryotic cells, each of which is composed of four subunits, which are about 20–40% related to each other.
AP-1 and AP-2 are involved in sorting proteins into clathrin-coated vesicles (CCVs), while AP-3 and AP-4 appear to be able to operate without clathrin. Importantly, each adaptor protein complex has a distinct localization and function, although all are involved in the post-Golgi transport of cellular cargo.
AP-1 trafficks proteins between tubular endosomes and the TGN. AP-2 facilitates clathrin-mediated endocytosis, AP-3 shuffles cargo from tubular endosomes to late endosomes, lysosomes, and related organelles, while AP-4 has recently been shown to traffic the amyloid precursor protein from the TGN to endosomes. Another more distantly related heterotetrameric complex is the F subcomplex of the COPI coat (F-COPI), which acts in an earlier pathway, by packaging cargo into vesicles for retrograde trafficking from the Golgi apparatus to the ER.
Subunits from all four AP complexes and COPI have been identified in genome sequences from a diverse range of eukaryotic organisms, suggesting they must have emerged over a billion years ago, the researchers note. Homology searches of available databases haven’t found any new family members, suggesting—until now—that all the AP complexes had been identified.
However, three other families of proteins contain μ homology domains (MHDs: μ1 or μ2 are subunits in AP-1 and AP-2, respectively) that are related to the C-terminal cargo-binding domains of AP μ subunits, and one of these sparked the interest of the Alberta and Cambridge teams. The protein is encoded by a human gene designated C14orf108, FLJ10813, or MUDENG (for μ-2-related death-inducing gene). A homolog of this gene is also present in Naegleria, an organism in the Excavata supergroup that split off from other eukaryotic lineages over a billion years ago. The researchers decided to characterize C14orf108 further and identify its binding partners, localization, and function.
Initial analyses showed that the protein encoded by the C14orf108 gene is 490 amino acids long with a predicted molecular weight of 54.7 kD, which is similar in size to the μ-adaptin subunits in AP-1 and AP-2 and also to the δ-COP subunit of F-COPI. Structural predictions indicated that the secondary structures of both the C-terminal and N-terminal of C14orf108 are also similar to those of the μ-adaptins and δ-COP.
This is in contrast to the other two families of MHD-containing proteins, which have completely different N-termain domains, the authors note. “These observations suggested to us that C14orf108 might be the μ subunit of a new, previously unsuspected AP complex,” they write.
Screening a human placental yeast two-hybrid library for potential binding partners for C14orf108 highlighted one previously uncharacterized sequence, derived from a gene encoding a protein called DKFZp761E198. All the top hits resulting from a subsequent analysis of the protein’s sequence using an iterative PSI-BALST search were β-adaptins.
This further suggested that, like the μ-adaptins and δ-COP, C14orf108 interacts with a β-adaptin-related protein. Supporting this, secondary structure predictions reveal that DKFZp761E198 is structurally very similar to a β-adaptin or β-COP and includes a binding site consistent with a β-μ-like interaction.
The next step was to see where C14orf108 is distributed in cells, using antibodies against the protein. The results showed that, like clathrin and AP-1, C14orf108 was found both in membranes and in the cytosol at approximately equal amounts, which indicated that it cycles on and off the membrane.
Unlike clathrin and AP-1, however, it wasn’t found in clathrin-coated vesicles (CCV). Further analysis to home in on the protein suggested that it localized at late endosomes and lysosomes, suggesting it may function in the late endocytic pathway.
To find out more about the function of C14orf108, the researchers knocked out endogenous protein from cells using siRNA and looked for defects in endocytic trafficking. They found that the knockout cells exhibited a marked change in localization of the cation-independent mannose 6-phosphate receptor (CIMPR), a receptor for lysosomal hydrolases that cycles between the TGN and endosomes. Knocking down AP-5 essentially caused the cell to form swollen endosomal structures with emanating tubules.
Similar results were observed when siRNAs were used to knock down DKFZp761E198, which the team suggests provides evidence that the interaction between C14orf and DKFZp761E198 is relevant to function. These localization results, combined with data from conventional and immunogold electron microscopy, further supported a role for the C14orf108-DKFZp761E198 complex in endosomal trafficking.
The team looked for the C14orf108-DKFZp761E198 complex in another 29 eukaryotic genomes spanning the five major supergroups. Candidate sequences were classified as homologues of subunits of one of the four known AP complexes or of the novel complex. This approach identified β-adaptin subunit homologs of DKFZp761E198 and similarly μ-adaptin subunit homologues of C14orf108.
The overall evidence was thus heavily weighted in favor of C14orf108 and DKFZp761E198 representing bona fide AP complex subunits: “Therefore, we suggest that C14orf108 and DKFZp761E198 should be renamed μ5 and β5, respectively, and that the complex they form should be called AP-5,” the authors conclude.
In fact, candidate homologues were identified for μ5 and β5 in at least two representatives from each of the five major eukaryotic supergroups, which indicated that AP-5 is broadly conserved in eukaryotes but has been lost through divergence. “Most excitingly, analysis of the concatenated β and μ dataset, with the COP sequences used as an outgroup, provided the first robust evolutionary order of emergence for the adaptin complexes,” they continue.
“Taking into account previous data showing that AP-1 and AP-2 were the most recent complexes to evolve, we can now hypothesize that AP-3 is the basal adaptor complex, followed by AP-5, AP-4, and then finally AP-1 and AP-2.”
Given that the four AP complexes and the F-COPI complex are all heterotetramers, it was reasonable to suggest that AP-5 also has a heterotetrameric structure, comprising μ5 and β5, and another, yet to be identified large subunit and small subunit. Unfortunately, the authors’ yeast two-hybrid screens and PSI-BLAST/HHMer searches all failed to identify candidates, and they weren’t able to carry out co-immunoprecipitation studies.
Instead, they were drawn to recent independent research that had identified both DKFZp761E198/β5 and C14orf108/μ5 along with another uncharacterised protein, C20orf29, in immunoprecipitations carried out using tagged versions of three proteins called KIAA0415, SPG11, and SPG15. KIAA0415 was found in an RNAi library screen for genes involved in DNA repair, and SPG11 and SPG15 are mutated in hereditary spastic paraplegia (HSP). The authors of this research also provided evidence that KIAA0415 is similarly mutated in some patients with HSP.
A number of features of KIAA0415/SPG48 and C20orf29 suggested that they could be the large subunit and the small subunit of the AP-5 complex, the researchers state. The proteins were the right size, KIAA0415/SPG48 displayed a similar predicted secondary structure to the known AP complex subunits γ/α/δ/ε, and C20orf29 had a similar predicted secondary structure to the σ subunits.
In addition, iterative PSI-BLAST and HHpred searches using KIAA0415/SPG48 and C20orf29 identified AP large subunits and small subunits, respectively, as principal homologues, while homologues of both KIAA0415/SPG48 and C20orf29 were found in nearly all the taxa that also possessed C14orf108/μ5 and DKFZp761E198/β5. Separate studies confirmed that knocking down either KIAA0415/SPG48 or C20orf29 produced the same phenotype as knocking down μ5 or β5.
Fortunately, the team was able to immunoprecipitate GFP-tagged C20orf29 and then probe Western blots with antibodies against the other subunits. These results provided independent confirmation that proteins that were detected in immunoprecipitates with antibodies against tagged KIAA0415 also precipitated with antibodies against DKFZp761E198/β5.
“Thus, we propose that like the other AP complexes, AP-5 is a heterotetramer, consisting of two large subunits: a medium subunit and a small subunit ,” the authors conclude. “We also propose that like other AP complexes, AP-5 binds accessory proteins, two of which are SPG11 and SPG15.”
The evidence pointing to the late endosome and/or lysosome as the site of action of AP-5 is interesting as trafficking out of late endosomes has never been formally established, although there are a number of late endosomal membrane proteins that do need to be recycled, the team adds. “The connection with hereditary spastic paraplegia, a group of genetic disorders that already have a number of links with membrane traffic, provides a promising lead for future investigations into AP-5 function,” they stress.
That AP-5 hasn’t previously been identified and may relate in part to the fact that it isn’t present in a number of the major model organisms used to study membrane trafficking, including Saccharomyces cerevisiae. AP-5’s similarity to the rest of the AP family is also relatively weak.
“Even when comparing the same AP-5 subunit in closely related species like humans and mice, the degree of conservation is surprisingly low,” Dr. Dacks et al. point out. For example, while human and mouse β2 are 99.9% identical, human and mouse β5 are only 85% identical.
“More generally, as seen from the phylogenetic analyses, the AP-5 components always represent divergent sequences, pointing to a lack of selective pressure on AP-5, which has made the subunits very difficult to identify even with sophisticated bioinformatic techniques.”