Novel insights into plant light-harvesting complex II phosphorylation and 'state transitions'

Plants need a highly responsive regulatory system to keep photosynthetic light reactions in balance with the needs and restrictions of the downstream metabolism. This mechanism optimises plant growth under naturally fluctuating light conditions. In this opinion article, we present a model addressing the biological role of the light intensity-controlled phosphorylation of light-harvesting complex II (LHCII) proteins and its relation with the non-photochemical quenching of excitation energy (NPQ). We overturn a long held view of the possible role of 'state transitions'. Instead, we discuss the interplay between LHCII protein phosphorylation and NPQ, a mechanism that is crucial for regulating excitation energy distribution to the two photosystems (PSII and PSI) and balancing the intersystem electron flow despite constant fluctuations in light intensity.     

Single-molecule electron tunneling spectroscopy of the higher plant light-harvesting complex LHC II

Electronic spectroscopy of a single biological molecule is demonstrated with approximately 4 A spatial resolution. The light-harvesting complex II (LHC II), in the ground and photo-excited states, was studied using scanning tunneling microscopy and spectroscopy of intact Photosystem II complexes. Analysis of the spectra indicates that the main mechanisms of tunneling between the STM tip and the surface involve delocalized electronic states of the LHC II and local vibronic states associated with C=C, C=O, C-H, N-H, and O-H groups near the LHC II surface. Conduction within the bulk LHC II is then due to ohmic and hopping conduction as well as tunneling between amino acid residues. Light activation of LHC II occurs via a photoconductive rather than a photovoltaic mechanism. There is a dramatic light-induced increase in the electronic density of states indicating a light-induced enhancement of energy and electron delocalization which is important for the efficient and rapid transfer of excitation energy from LHC II to the Photosystem II reaction center.     

Three-dimensional electron diffraction of plant light-harvesting complex

Electron diffraction patterns of two-dimensional crystals of light-harvesting chlorophyll a/b-protein complex (LHC-II) from photosynthetic membranes of pea chloroplasts, tilted at different angles up to 60°, were collected to 3.2 Å resolution at -125°C. The reflection intensities were merged into a three-dimensional data set. The Friedel R-factor and the merging R-factor were 21.8 and 27.6%, respectively. Specimen flatness and crystal size were critical for recording electron diffraction patterns from crystals at high tilts. The principal sources of experimental error were attributed to limitations of the number of unit cells contributing to an electron diffraction pattern, and to the critical electron dose. The distribution of strong diffraction spots indicated that the three-dimensional structure of LHC-II is less regular than that of other known membrane proteins and is not dominated by a particular feature of secondary structure.     

Evidence for the role of the light-harvesting chlorophyll protein complex in photosystem II heterogeneity

Analyses of chlorophyll fluorescence induction kinetics from DCMU-poisoned thylakoids were used to examine the contribution of the light-harvesting chlorophyll protein complex (LHCP) to Photosystem II (PS II) heterogeneity. Thylakoids excited with 450 nm radiation exhibited fluorescence induction kinetics characteristic of major contributions from both PS II_α and PS II_β centres. On excitation at 550 nm the major contribution was from PS II_β centres, that from PS II_α centres was only minimal. Mg²⁺ depletion had negligible effect on the induction kinetics of thylakoids excited with 550 nm radiation, however, as expected, with 450 nm excitation a loss of the PS II_α component was observed. Thylakoids from a chlorophyll-b-less barley mutant exhibited similar induction kinetics with 450 and 550 nm excitation, which were characteristic of PS II_β centres being the major contributors; the PS II_α contribution was minimal. The fluorescence induction kinetics of wheat thylakoids at two different developmental stages, which exhibited different amounts of thylakoid appression but similar chlorophyll ratios and thus similar PS II:LHCP ratios, showed no appreciable differences in the relative contributions of PS II_α and PS II_β centres. Mg²⁺ depletion had similar effects on the two thylakoid preparations. These data lead to the conclusion that it is the PS II:LHCP ratio, and probably not thylakoid appression, that is the major determinant of the relative contributions of PS II_α and PS II_β to the fluorescence induction kinetics. PS II_α characteristics are produced by LHCP association with PS II, whereas PS II_β characteristic can be generated by either disconnecting LHCP from PS II or by preferentially exciting PS II relative to LHCP.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-β,d-maltoside, n-octyl-β,d-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

How the molecular structure determines the flow of excitation energy in plant light-harvesting complex II

Excitation energy transfer in the light-harvesting complex II of higher plants is modeled using excitonic couplings and local transition energies determined from structure-based calculations recently (Müh et al., 2010). A theory is introduced that implicitly takes into account protein induced dynamic localization effects of the exciton wavefunction between weakly coupled optical and vibronic transitions of different pigments. Linear and non-linear optical spectra are calculated and compared with experimental data reaching qualitative agreement. High-frequency intramolecular vibrational degrees of freedom are found important for ultrafast subpicosecond excitation energy transfer between chlorophyll (Chl) b and Chla, since they allow for fast dissipation of the excess energy. The slower ps component of this transfer is due to the monomeric excited state of Chlb 605. The majority of exciton relaxation in the Chla spectral region is characterized by slow ps exciton equilibration between the Chla domains within one layer and between the lumenal and stromal layers in the 10-20 ps time range. Subpicosecond exciton relaxation in the Chla region is only found within the terminal emitter domain (Chls a 610/611/612) and within the Chla 613/614 dimer. Deviations between measured and calculated exciton state life times are obtained for the intermediate spectral region between the main absorbance bands of Chla and Chlb that indicate that besides Chlb 608 another pigment should absorb there. Possible candidates, so far not identified by structure-based calculations, but by fitting of optical spectra and mutagenesis studies, are discussed. Additional mutagenesis studies are suggested to resolve this issue.     

Importance of trimer-trimer interactions for the native state of the plant light-harvesting complex II

Aggregates and solubilized trimers of LHCII were characterized by circular dichroism (CD), linear dichroism and time-resolved fluorescence spectroscopy and compared with thylakoid membranes in order to evaluate the native state of LHCII in vivo. It was found that the CD spectra of lamellar aggregates closely resemble those of unstacked thylakoid membranes whereas the spectra of trimers solubilized in n-dodecyl-beta,D-maltoside, n-octyl-beta,D-glucopyranoside, or Triton X-100 were drastically different in the Soret region. Thylakoid membranes or LHCII aggregates solubilized with detergent exhibited CD spectra similar to the isolated trimers. Solubilization of LHCII was accompanied by profound changes in the linear dichroism and increase in fluorescence lifetime. These data support the notion that lamellar aggregates of LHCII retain the native organization of LHCII in the thylakoid membranes. The results indicate that the supramolecular organization of LHCII, most likely due to specific trimer-trimer contacts, has significant impact on the pigment interactions in the complexes.     

Diffusion of light-harvesting complex II in the thylakoid membranes

The light-harvesting complex II (LHCII) is the main energy absorber for photosynthesis in green plants, and its translocation between photosystems I and II is the primary means of energy redistribution between them. Using single-particle tracking, we performed the first measurement of the mobility of LHCII in the photosynthetic membranes in both the nonphosphorylated and the phosphorylated (P-LHCII) conformations. These are part of an important, reversible, energy re-equilibration process called the state transition. We found that the population of P-LHCII in unappressed membranes is more mobile than the population of non-P-LHCII from the same regions.     

Supercomplexes of plant photosystem I with cytochrome b6f,light-harvesting complex II and NDH

Photosystem I (PSI) is a pigment-protein complex required for the light-dependent reactions of photosynthesis and participates in light-harvesting and redox-driven chloroplast metabolism. Assembly of PSI into supercomplexes with light harvesting complex (LHC) II, cytochrome b₆f (Cytb₆f) or NAD(P)H dehydrogenase complex (NDH) has been proposed as a means for regulating photosynthesis. However, structural details about the binding positions in plant PSI are lacking. We analyzed large data sets of electron microscopy single particle projections of supercomplexes obtained from the stroma membrane of Arabidopsis thaliana. By single particle analysis, we established the binding position of Cytb₆f at the antenna side of PSI. The rectangular-shaped Cytb₆f dimer binds at the side where Lhca1 is located. The complex binds with its short side rather than its long side to PSI, which may explain why these supercomplexes are difficult to purify and easily disrupted. Refined analysis of the interaction between PSI and the NDH complex indicates that in total up to 6 copies of PSI can arrange with one NDH complex. Most PSI-NDH supercomplexes appeared to have 1–3 PSI copies associated. Finally, the PSI-LHCII supercomplex was found to bind an additional LHCII trimer at two positions on the LHCI side in Arabidopsis. The organization of PSI, either in a complex with NDH or with Cytb₆f, may improve regulation of electron transport by the control of binding partners and distances in small domains.     

Refined structure-based simulation of plant light-harvesting complex II: Linear optical spectra of trimers and aggregates

Linear optical spectra of solubilized trimers and small lamellar aggregates of the major light-harvesting complex II (LHCII) of higher plants are simulated employing excitonic couplings and site energies of chlorophylls (Chls) computed on the basis of the two crystal structures by a combined quantum chemical/electrostatic approach. A good agreement between simulation and experiment is achieved (except for the circular dichroism in the Chl b region), if vibronic transitions of Chls are taken into account. Site energies are further optimized by refinement fits of optical spectra. The differences between refined and directly calculated values are not significant enough to decide, whether the crystal structures are closer to trimers or aggregates. Changes in the linear dichroism spectrum upon aggregation are related to site energy shifts of Chls b601, b607, a603, a610, and a613, and are interpreted in terms of conformational changes of violaxanthin and the two luteins involving their ionone rings. Chl a610 is the energy sink at 77K in both conformations. An analysis of absorption spectra of trimers perpendicular and parallel to the C(3)-axis (van Amerongen et al. Biophys. J. 67 (1994) 837-847) shows that only Chl a604 close to neoxanthin is significantly reoriented in trimers compared to the crystal structures. Whether this pigment is orientated in aggregates as in the crystal structures, can presently not be determined faithfully. To finally decide about pigment reorientations that could be of relevance for non-photochemical quenching, further polarized absorption and fluorescence measurements of aggregates or detergent-depleted LHCII would be helpful. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Light-induced excitation quenching and structural transition in light-harvesting complex II

Light-induced fluorescence quenching of chlorophyll a in light-harvesting complex II (LHCII) incorporated into liposomes was examined. The rate of fluorescence quenching was found to depend on the incubation temperature. The effect was almost not observed at liquid nitrogen temperature, demonstrated a lag phase after onset of light at temperatures below 25 °C and was most distinctly pronounced at temperatures above 25 °C. Energetic uncoupling of accessory xanthophylls and chlorophyll a, and energetic uncoupling of chlorophyll b and chlorophyll a were observed as accompanying the excitation quenching. The observed changes were reversible during dark incubation. Similar energetic uncoupling was also observed in darkness, induced by the increase in temperature. Additionally, the temperature characteristics of fluorescence measurements displayed a pronounced transition in the region of 22–25 °C. The experiments carried out with the monomolecular layer technique in dicated a structural transition of LHC II in the same temperature region as demonstrated by an increase in the mean molecular area of LHC II at the argon-water interface. Alterations in surface topography induced by temperature changes could also be observed with scanning force microscopy of LHC II monolayers deposited as Langmuir-Blodgett films onto glass slides. The transition was found to be associated with the enhanced excitation energy consumption by the protein, monitored by calorimetric measurements. It is proposed that the observed transition efficiently protects LHC II against overexcitation-related damage and is therefore of physiological importance.     

Assembly of the major light-harvesting complex II in lipid nanodiscs

Self-aggregation of isolated plant light-harvesting complexes (LHCs) upon detergent extraction is associated with fluorescence quenching and is used as an in vitro model to study the photophysical processes of nonphotochemical quenching (NPQ). In the NPQ state, in vivo induced under excess solar light conditions, harmful excitation energy is safely dissipated as heat. To prevent self-aggregation and probe the conformations of LHCs in a lipid environment devoid from detergent interactions, we assembled LHCII trimer complexes into lipid nanodiscs consisting of a bilayer lipid matrix surrounded by a membrane scaffold protein (MSP). The LHCII nanodiscs were characterized by fluorescence spectroscopy and found to be in an unquenched, fluorescent state. Remarkably, the absorbance spectra of LHCII in lipid nanodiscs show fine structure in the carotenoid and Q_y region that is different from unquenched, detergent-solubilized LHCII but similar to that of self-aggregated, quenched LHCII in low-detergent buffer without magnesium ions. The nanodisc data presented here suggest that 1), LHCII pigment-protein complexes undergo conformational changes upon assembly in nanodiscs that are not correlated with downregulation of its light-harvesting function; and 2), these effects can be separated from quenching and aggregation-related phenomena. This will expand our present view of the conformational flexibility of LHCII in different microenvironments.     

Excitonic coupling of chlorophylls in the plant light-harvesting complex LHC-II.

Manifestation and extent of excitonic interactions in the red Chl-absorption region (Q(y) band) of trimeric LHC-II were investigated using two complementary nonlinear laser-spectroscopic techniques. Nonlinear absorption of 120-fs pulses indicates an increased absorption cross section in the red wing of the Q(y) band as compared to monomeric Chl a in organic solution. Additionally, the dependence of a nonlinear polarization response on the pump-field intensity was investigated. This approach reveals that one emitting spectral form, characterized by a 2.3(+/-0.8)-fold larger dipole strength than monomeric Chl a, dominates the fluorescence spectrum of LHC-II. Considering available structural and spectroscopic data, these results can be consistently explained assuming the existence of an excitonically coupled dimer located at Chl-bindings sites a2 and b2 (referring to the original notation of W. Nühlbrandt, D.N. Wang, and Y. Fujiyoshi, Nature, 1994, 367:614-621), which must not necessarily correspond to Chls a and b). This fluorescent dimer, terminating the excitation energy-transfer chain of the LHC-II monomeric subunit, is discussed with respect to its relevance for intra- and inter-antenna excitation energy transfer.     

Degradation of the main Photosystem II light-harvesting complex.

Many factors trigger the degradation of proteins, including changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Despite the importance for viability, still very little is known about protein degradation and its regulation. The degradation of the most abundant membrane protein on Earth, the light-harvesting complex of Photosystem II (LHC II), is highly regulated under different environmental conditions, e.g. light stress, to prevent photochemical damage of the reaction center. However, despite major effort to identify the protease/proteases involved in the degradation of the apoproteins of LHC II the molecular details of this important process remain obscure. LHC II belongs to the family of chlorophyll a/b binding proteins (CAB proteins) and is located in the thylakoid membrane of the plant chloroplast. The results of biochemical experiments to isolate and characterize the protease degrading LHC II are summarized here and compared to our own recent finding indicating that a metalloprotease of the FtsH family is involved in this process.     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis     

Crystallisation, structure and function of plant light-harvesting Complex II

The chlorophyll a/b light-harvesting complex of photosystem II (LHC-II) collects most of the solar energy in the biosphere. LHC-II is the prototype of a highly conserved family of membrane proteins that fuels plant photosynthesis in the conversion of excitation energy into biologically useful chemical energy. In addition, LHC-II plays an important role in the organisation of the thylakoid membrane, the structure of the photosynthetic apparatus, the regulation of energy flow between the two photosystems, and in the controlled dissipation of excess excitation energy under light stress. Our current understanding of the sophisticated mechanisms behind each of these processes has profited greatly from the progress made over the past two decades in determining the structure of the complex. This review presents the developments and breakthroughs that ultimately lead to the high-resolution structure of LHC-II. Based on an alignment of the remarkably well engineered and highly conserved LHC polypeptide, we propose several key features of the LHC-II structure that are likely to be present in all members of the LHC family. Finally, some recently proposed mechanisms of energy-dependent non-photochemical quenching (NPQ) are examined from a structural perspective.     

Stark spectroscopy of the light-harvesting complex II in different oligomerisation states

The electric field-induced absorption changes (Stark effect) of light-harvesting complex II (LHCII) in different oligomerisation states-monomeric, trimeric and aggregated-have been probed at 77 K. All the chlorophyll (Chl) a molecules exhibit electro-optic properties in the Q(y) absorption region characterized by a change in dipole moment /Deltamu-->/ =0.6+/-0.06D/f and polarizability, Tr(Deltaalpha;) approximately 55+/-5 A(3)/f(2) upon electronic excitation, which are similar to those of unbound monomeric Chl a, indicating the absence of strong delocalization of the excitations which would be expected in the presence of strong excitonic interactions. The Stark effect in the Chl b absorption region is significantly bigger with /Deltamu-->/ values of the order of 2.0+/-0.2 D/f and it is attributed to strong interactions with neoxanthin molecules. Clear oligomerisation-dependent differences are observed in the carotenoid region, mainly due to the appearance of a new xanthophyll absorption band at 509 in the spectra of trimers and oligomers. It is ascribed to some lutein molecules, in agreement with previous experimental observations. The electro-optic properties of these lutein molecules are significantly different from those of the other xanthophylls in LHCII, which do not exhibit such a big change in dipole moment upon electronic excitation (/Deltamu-->/ =14.6+/-2.0 D/f). Upon aggregation of LHCII some extra absorption appears on the red side of the main Chl a Q(y) absorption band. In contrast to an earlier suggestion [J. Phys. Chem., A 103 (1999) 2422], no indications are found for the charge-transfer character of the corresponding band. The assignments of the S(2) electronic transitions of neoxanthin and lutein in LHCII and possible origins of the Stark effect are discussed.     

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Eukaryotic cells have membrane-enclosed organelles, which carry out specialized functions, including compartmentalized biochemical reactions, metabolic channeling, and regulated signaling, inside a single cell. The transport of proteins, lipids, and other molecules between these organelles is mediated largely by small vesicular carriers that bud off at a donor compartment and fuse with the target membrane to deliver their cargo. The generation of these vesicles has been subject to extensive studies and has led to the identification of numerous coat proteins that are required for their formation at different sites (1, 2). Coat proteins can be monomers, but in most cases, they consist of several proteins, which form a heteromeric complex.Heterotetrameric adapter protein (AP) complexes are required at several endomembranes for cargo binding. Five well-conserved AP-complexes with differing functions have been identified in mammalian cells, named AP-1–AP-5, of which three (AP-1–AP-3) are conserved from yeast to human (3, 4). The three conserved adapter complexes function at different membranes along the endomembrane system. AP-1 is required for cargo transport between the Golgi and the endosome, AP-2 is required for cargo recognition and transport between the plasma membrane and the early endosome. Finally, AP-3 functions between the trans Golgi and the vacuole in yeast, whereas mammalian AP-3 localizes to a tubular endosomal compartment, in addition to or instead of the TGN (2, 5, 6).Each of the complexes consists of four different subunits: two large adaptins (named α−ζ and β1-5 respectively), a medium-sized subunit (μ1-5), and a small subunit (σ1-5). While μ- and σ-subunits together with the N-termini of the large adaptins build the membrane-binding core of the complex, the C-termini of both adaptins contain the ear domains, which are connected via flexible linkers (2). The recruitment of these complexes to membranes is not entirely conserved. They all require cargo binding, yet AP-1 binds Arf1-GTP with the γ and β1 subunit and phosphatidylinositol-4-phosphate (PI4P) via a proposed conserved site on its γ-subunit (7, 8). AP-2, on the other hand, interacts with PI(4,5)P₂ at the plasma membrane via its α, β2, and μ2 subunits (9, 10, 11).Several studies have uncovered how AP-3 functions in cargo sorting in yeast. AP-3 recognizes cargo at the Golgi via two sorting motifs in the cytosolic segments of membrane proteins: a Yxxφ sorting motif, as found in yeast in the SNARE Nyv1 or the Yck3 casein kinase, which binds to a site in μ3, as shown for mammalian AP-3, which is similar to μ2 in AP-2 (12, 13, 14), and dileucine motifs as found in the yeast SNARE Vam3 or the alkaline phosphatase Pho8, potentially also at a site comparable to AP-1 and AP-2 (15, 16). Unlike AP-1 and AP-2-coated vesicles, which depend on clathrin for their formation (2, 17), AP-3 vesicle formation in yeast does not require clathrin or the HOPS subunit Vps41 (18), yet Vps41 is required at the vacuole to bind AP-3 vesicles prior to fusion (19, 20, 21, 22). Studies in metazoan cells revealed that Vps41 and AP-3 function in regulated secretion (23, 24, 25), and AP-3 is required for biogenesis of lysosome-related organelles (26). This suggests that the AP-3 complex has features that are quite different from AP-1 and AP-2 complexes, which cooperate with clathrin in vesicle formation (2).Among the three conserved AP complexes, the function of the AP-3 complex is the least understood. Arf1 is necessary for efficient AP-3 vesicle generation in mammalian cells and shows a direct interaction with the β3 and δ subunits of AP-3 (27, 28). In addition, in vitro experiments on mammalian AP-3 using liposomes or enriched Golgi membranes suggest Arf1 as an important factor in AP-3 recruitment, whereas acidic lipids do not have a major effect, in contrast to what was found for AP-1 and AP-2 (7, 11, 29, 30). Another study showed that membrane recruitment of AP-3 depends on the recognition of sorting signals in cargo tails and PI3P (31), similar to AP-1 recruitment via cargo tails, Arf1 and PI4P (32).However, since AP-1 and AP-3 are both recruited to the trans-Golgi network (TGN) in yeast (33), the mechanism of their recruitment likely differs. Even though Arf1 is required, yeast AP-3 seems to be present at the TGN before the arrival of the Arf1 guanine nucleotide exchange factor (GEF) Sec7 (33). This implies the necessity for additional factors at the TGN and a distinct mechanism to allow for spatial and temporal separation of AP-1 and AP-3 recruitment to membranes. Structural data on mammalian AP-1 and AP-2 “core” complexes without the hinge and ear domains of their large subunits revealed that both exist in at least two very defined conformational states: a “closed” cytosolic state, where the cargo-binding sites are buried within the complex, and an “open” state, where the same sites are available to bind cargo (7, 8, 10, 34, 35). Binding of Arf1 to AP-1 or PI(4,5)P₂ in case of AP-2 induces a conformational change in the complexes that enables them to bind cargo molecules carrying a conserved acidic di-Leucine or a Tyrosine-based motif, as for all three AP complexes in yeast (8, 34). Additional conformational states and intermediates have been reported for both, mammalian AP-1 and AP-2 complex. AP-1, for example, can be hijacked by the human immunodeficiency virus-1 (HIV-1) proteins viral protein u (Vpu) and negative factor (Nef), resulting in a hyper-open conformation of AP-1 (36, 37).An emerging model over the past years has suggested that APs have several binding sites that allow for the stabilization of membrane binding and the open conformation of the complexes, but there are initial interactions required that dictate their recruitment to the target membrane. Although these interaction sites for mammalian AP-1 and AP-2 have been identified in great detail based on interaction analyses and structural studies (8, 10, 11, 35, 36, 38, 39), structural data for AP-3 is largely missing. The C-terminal part of the μ-subunit of mammalian AP-3 has been crystallized together with a Yxxφ motif-containing a cargo peptide, which revealed a similar fold and cargo-binding site as shown for AP-1 and AP-2 (14). However, positively charged binding surfaces required for PIP-interaction were not well conserved. Although the “trunk” segment of AP-1 and AP-2 is known quite well by now, information on hinge and ear domains in context of these complexes is largely missing. Crystal structures of the isolated ear domains of α-, γ- and β2-adaptin have been published (40, 41, 42), and a study on mammalian AP-3 suggested a direct interaction between δ-ear and δ3 that interfered with Arf1-binding (43). Furthermore, during tethering of AP-3 vesicles with the yeast vacuole, the δ−subunit Apl5 of the yeast AP-3 complex binds to the Vps41 subunit of the HOPS complex as a prerequisite of fusion (18, 19, 21, 22).In this study, we applied single particle electron cryo-microscopy (cryo-EM) to analyze the purified full-length AP-3 complex from yeast and unraveled the factors required for AP-3 recruitment to membranes by biochemical reconstitution. Our data reveal that a surprisingly flexible AP-3 complex requires a combination of cargo, PI4P, and Arf1 for membrane binding, which explains its function in selective cargo sorting at the Golgi.