The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches.

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin-binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin-binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

Adaptor protein (AP) complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

Autosomal recessive spastic paraplegia 52 is caused by biallelic mutations in AP4S1 which encodes a subunit of the adaptor protein complex 4 (AP-4). Using next-generation sequencing, we identified three novel unrelated SPG52 patients from a cohort of patients with cerebral palsy. The discovered variants in AP4S1 lead to reduced AP-4 complex formation in patient-derived fibroblasts. To further understand the role of AP4S1 in neuronal development and homeostasis, we engineered the first zebrafish model of AP-4 deficiency using morpholino-mediated knockdown of ap4s1. In this model, we discovered several phenotypes mimicking SPG52, including altered CNS development, locomotor deficits, and abnormal neuronal excitability.

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

GGAs (Golgi-localised, γ-ear containing, ADP ribosylation factor-binding) are a family of clathrin adaptors that sort a number of biologically important transmembrane proteins into clathrin-coated vesicles. Knockout and knockdown studies to determine GGA function are confounded by the fact that there are 3 GGA genes in mammalian cells. Thus Drosophila melanogaster is a useful model system to study tissue expression profiles and knockdown phenotypes as there is a single GGA ortholog.

Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions.

Adaptor protein complex 5 (AP-5) and its partners, SPG11 and SPG15, are recruited onto late endosomes and lysosomes. Here we show that recruitment of AP-5/SPG11/SPG15 is enhanced in starved cells and occurs by coincidence detection, requiring both phosphatidylinositol 3-phosphate (PI3P) and Rag GTPases. PI3P binding is via the SPG15 FYVE domain, which, on its own, localizes to early endosomes. GDP-locked RagC promotes recruitment of AP-5/SPG11/SPG15, while GTP-locked RagA prevents its recruitment. Our results uncover an interplay between AP-5/SPG11/SPG15 and the mTORC1 pathway and help to explain the phenotype of AP-5/SPG11/SPG15 deficiency in patients, including the defect in autophagic lysosome reformation.

Adaptor protein complex 4-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of adaptor protein complex 4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here, we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The 'ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 ± 0.13 versus 1.21 ± 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia.

The AP-5 complex is a recently identified but evolutionarily ancient member of the family of heterotetrameric adaptor proteins (AP complexes). It is associated with two proteins that are mutated in patients with hereditary spastic paraplegia, SPG11 and SPG15. Here we show that the four AP-5 subunits can be coimmunoprecipitated with SPG11 and SPG15, both from cytosol and from detergent-extracted membranes, with a stoichiometry of ∼1:1:1:1:1:1. Knockdowns of SPG11 or SPG15 phenocopy knockdowns of AP-5 subunits: all six knockdowns cause the cation-independent mannose 6-phosphate receptor to become trapped in clusters of early endosomes. In addition, AP-5, SPG11, and SPG15 colocalize on a late endosomal/lysosomal compartment. Both SPG11 and SPG15 have predicted secondary structures containing α-solenoids related to those of clathrin heavy chain and COPI subunits. SPG11 also has an N-terminal, β-propeller-like domain, which interacts in vitro with AP-5. We propose that AP-5, SPG15, and SPG11 form a coat-like complex, with AP-5 involved in protein sorting, SPG15 facilitating the docking of the coat onto membranes by interacting with PI3P via its FYVE domain, and SPG11 (possibly together with SPG15) forming a scaffold.

Adaptor proteins (AP 1-5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and 'fingerprint bodies'. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs.

We developed "fractionation profiling," a method for rapid proteomic analysis of membrane vesicles and protein particles. The approach combines quantitative proteomics with subcellular fractionation to generate signature protein abundance distribution profiles. Functionally associated groups of proteins are revealed through cluster analysis. To validate the method, we first profiled >3500 proteins from HeLa cells and identified known clathrin-coated vesicle proteins with >90% accuracy. We then profiled >2400 proteins from Drosophila S2 cells, and we report the first comprehensive insect clathrin-coated vesicle proteome. Of importance, the cluster analysis extends to all profiled proteins and thus identifies a diverse range of known and novel cytosolic and membrane-associated protein complexes. We show that it also allows the detailed compositional characterization of complexes, including the delineation of subcomplexes and subunit stoichiometry. Our predictions are presented in an interactive database. Fractionation profiling is a universal method for defining the clathrin-coated vesicle proteome and may be adapted for the analysis of other types of vesicles and particles. In addition, it provides a versatile tool for the rapid generation of large-scale protein interaction maps.

Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the "knocksideways" system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins.

We report identical twins with intellectual disability, progressive spastic paraplegia and short stature, born to a consanguineous family. Intriguingly, both children presented with lymphadenitis caused by the live Bacillus Calmette-Guérin (BCG) vaccine. Two syndromes - hereditary spastic paraplegia (HSP) and mycobacterial disease - thus occurred simultaneously. Whole-exome sequencing (WES) revealed a homozygous nonsense mutation (p.R1105X) of the AP4E1 gene, which was confirmed by Sanger sequencing. The p.R1105X mutation has no effect on AP4E1 mRNA levels, but results in lower levels of AP-4ε protein and of the other components of the AP-4 complex, as shown by western blotting, immunoprecipitation and immunofluorescence. Thus, the C-terminal part of the AP-4ε subunit plays an important role in maintaining the integrity of the AP-4 complex. No abnormalities of the IL-12/IFN-γ axis or oxidative burst pathways were identified. In conclusion, we identified twins with autosomal recessive AP-4 deficiency associated with HSP and mycobacterial disease, suggesting that AP-4 may play important role in the neurological and immunological systems.

The GGAs [Golgi-localised, gamma-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.

Coat proteins are recruited onto membranes to form vesicles that transport cargo from one compartment to another, but the extent to which the cargo helps to recruit the coat proteins is still unclear. Here we have examined the role of cargo in the recruitment of Golgi-localized, gamma-ear-containing, ADP ribosylation factor (ARF)-binding proteins (GGAs) onto membranes in HeLa cells. Moderate overexpression of CD8 chimeras with cytoplasmic tails containing DXXLL-sorting signals, which bind to GGAs, increased the localization of all three GGAs to perinuclear membranes, as observed by immunofluorescence. GGA2 was also expressed at approximately twofold higher levels in these cells because it was degraded more slowly. However, this difference only partially accounted for the increase in membrane localization because there was a approximately fivefold increase in GGA2 associated with crude membranes and a approximately 12-fold increase in GGA2 associated with clathrin-coated vesicles (CCVs) in cells expressing CD8-DXXLL chimeras. The effect of cargo proteins on GGA recruitment was reconstituted in vitro using permeabilized control and CD8-DXXLL-expressing cells incubated with cytosol containing recombinant GGA2 constructs. Together, these results demonstrate that cargo proteins contribute to the recruitment of GGAs onto membranes and to the formation of GGA-positive CCVs.

The heterotetrameric AP and F-COPI complexes help to define the cellular map of modern eukaryotes. To search for related machinery, we developed a structure-based bioinformatics tool, and identified the core subunits of TSET, a 'missing link' between the APs and COPI. Studies in Dictyostelium indicate that TSET is a heterohexamer, with two associated scaffolding proteins. TSET is non-essential in Dictyostelium, but may act in plasma membrane turnover, and is essentially identical to the recently described TPLATE complex, TPC. However, whereas TPC was reported to be plant-specific, we can identify a full or partial complex in every eukaryotic supergroup. An evolutionary path can be deduced from the earliest origins of the heterotetramer/scaffold coat to its multiple manifestations in modern organisms, including the mammalian muniscins, descendants of the TSET medium subunits. Thus, we have uncovered the machinery for an ancient and widespread pathway, which provides new insights into early eukaryotic evolution.DOI: http://dx.doi.org/10.7554/eLife.02866.001.

Cerebral palsy due to perinatal injury to cerebral white matter is usually not caused by genetic mutations, but by ischemia and/or inflammation. Here, we describe an autosomal-recessive type of tetraplegic cerebral palsy with mental retardation, reduction of cerebral white matter, and atrophy of the cerebellum in an inbred sibship. The phenotype was recorded and evolution followed for over 20 years. Brain lesions were studied by diffusion tensor MR tractography. Homozygosity mapping with SNPs was performed for identification of the chromosomal locus for the disease. In the 14 Mb candidate region on chromosome 7q22, RNA expression profiling was used for selecting among the 203 genes in the area. In postmortem brain tissue available from one patient, histology and immunohistochemistry were performed. Disease course and imaging were mostly reminiscent of hypoxic-ischemic tetraplegic cerebral palsy, with neuroaxonal degeneration and white matter loss. In all five patients, a donor splice site pathogenic mutation in intron 14 of the AP4M1 gene (c.1137+1G-->T), was identified. AP4M1, encoding for the mu subunit of the adaptor protein complex-4, is involved in intracellular trafficking of glutamate receptors. Aberrant GluRdelta2 glutamate receptor localization and dendritic spine morphology were observed in the postmortem brain specimen. This disease entity, which we refer to as congenital spastic tetraplegia (CST), is therefore a genetic model for congenital cerebral palsy with evidence for neuroaxonal damage and glutamate receptor abnormality, mimicking perinatally acquired hypoxic-ischemic white matter injury.

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.