Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal enzymes tyrosinase-related protein 1 (Tyrp1) and tyrosinase follow an intracellular Golgi to melanosome pathway, whereas in the absence of glycosphingolipids, they are observed to pass over the cell surface. Unexpectedly, the lysosome-associated membrane protein 1 (LAMP-1) and 2 behaved exactly opposite: they were found to travel through the cell surface in control melanocytes but followed an intracellular pathway in the absence of glycosphingolipids. Chimeric proteins having the cytoplasmic tail of Tyrp1 or tyrosinase were transported like lysosomal proteins, whereas a LAMP-1 construct containing the lumenal domain of Tyrp1 localized to melanosomes. In conclusion, the lumenal domain contains sorting information that guides Tyrp1 and probably tyrosinase to melanosomes by an intracellular route that excludes lysosomal proteins and requires glucosylceramide.     

Lysosomal Targeting of Cystinosin Requires AP‐3

Cystinosin is a lysosomal cystine transporter defective in cystinosis, an autosomal recessive lysosomal storage disorder. It is composed of seven transmembrane (TM) domains and contains two lysosomal targeting motifs: a tyrosine‐based signal (GYDQL) in its C‐terminal tail and a non‐classical motif in its fifth inter‐TM loop. Using the yeast two‐hybrid system, we showed that the GYDQL motif specifically interacted with the μ subunit of the adaptor protein complex 3 (AP‐3). Moreover, cell surface biotinylation and total internal reflection fluorescence microscopy revealed that cystinosin was partially mislocalized to the plasma membrane (PM) in AP‐3‐depleted cells. We generated a chimeric CD63 protein to specifically analyze the function of the GYDQL motif. This chimeric protein was targeted to lysosomes in a manner similar to cystinosin and was partially mislocalized to the PM in AP‐3 knockdown cells where it also accumulated in the trans‐Golgi network and early endosomes. Together with the fact that the surface levels of cystinosin and of the CD63‐GYDQL chimeric protein were not increased when clathrin‐mediated endocytosis was impaired, our data show that the tyrosine‐based motif of cystinosin is a ‘strong’ AP‐3 interacting motif responsible for lysosomal targeting of cystinosin by a direct intracellular pathway.      

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal compartment that also exhibits budding profiles positive for AP-1. AP-3 colocalizes with clathrin, but to a lesser extent than does AP-1. The AP-3- and AP-1-bearing tubular compartments contain endocytosed transferrin, transferrin receptor, asialoglycoprotein receptor, and low amounts of the cation-independent mannose 6-phosphate receptor and the lysosome-associated membrane proteins (LAMPs) 1 and 2. Quantitative analysis revealed that of these distinct cargo proteins, only LAMP-1 and LAMP-2 are concentrated in the AP-3-positive membrane domains. Moreover, recycling of endocytosed LAMP-1 and CD63 back to the cell surface is greatly increased in AP-3-deficient cells. Based on these data, we propose that AP-3 defines a novel pathway by which lysosomal membrane proteins are transported from tubular sorting endosomes to lysosomes.     

AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways

The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19°C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro . The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.     

Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex

The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.     

Foreword: The structural biology of membrane proteins

Membrane protein-protein interactions are important for regulation, targeting, and activity of proteins in membranes but are difficult to detect and analyse. This review covers current approaches to studying membrane protein interactions. In addition to standard biochemical and genetic techniques, the classic yeast nuclear two-hybrid system has been highly successful in identification and characterization of soluble protein-protein interactions. However, classic yeast two-hybrid assays do not work for membrane proteins because such yeast-based interactions must occur in the nucleus. Here, we highlight recent advances in yeast systems for the detection and characterization of eukaryote membrane protein-protein interactions. We discuss these implications for drug screening and discovery.     

The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.     

Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B-like BHLH proteins

In-depth analysis of protein-protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ([DE]Lx(2)[RK]x(3)Lx(6)Lx(3)R) as the structural basis for interaction between MYB and R/B-like BHLH proteins. The motif has successfully been used to predict new MYB/BHLH interactions for A. thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants. In A. thaliana, the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins. It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface-exposed pattern of hydrophobic and charged residues. Single-site mutation of any amino acid of the signature impairs the interaction. Two particular amino acids have been determined to account for most of the interaction stability. Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay. Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation. By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B-like BHLH proteins potentially involved in the TTG1-dependent regulatory interaction network. The results are discussed with respect to multi-functionality, specificity and redundancy of MYB and BHLH protein function.     

Production of crystallizable fragments of membrane proteins

Many membrane proteins feature autonomously folded extramembranous domains which, when isolated from the intact protein, perform biochemical functions relevant to biological activity. Whereas intact membrane proteins usually require detergent solubilization for purification, most extramembranous fragments are soluble in aqueous solution. If appropriately constructed, such fragments are often crystallizable and the resulting atomic structures can lead to important biological insight. In most instances, these fragments are produced in recombinant expression systems. To be crystallizable, molecular fragments should be uniform in composition and conformation and be available in abundance. Considerations for the production of crystallizable fragments of membrane proteins include the definition of fragment boundaries, the control of nonuniformities introduced by glycosylation or phosphorylation, and optimization of expression systems. These aspects are addressed here in general terms and in the case studies of applications to CD4, CD8, the insulin receptor kinase, and N-cadherin.     

Identification of a novel family of G protein-coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.     

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine‐based motif

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP‐fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild‐type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N‐terminal dileucine‐based motif. The NRAMP3 or NRAMP4 N‐terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4‐dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N‐terminal dileucine‐based motif as well as AP4.      

酵母双杂交筛选与果蝇C(2)M相互作用的蛋白

同源染色体联会时形成的联会复合体(Synaptonemal complex, SC)是由减数分裂前期Ⅰ多种蛋白质聚集而成的超级复合结构。生殖细胞特异性的核蛋白C(2)M(Crossover suppressor on 2 of Manheim)在染色体上高度聚集可以诱导SC的形成。本文采用酵母双杂交方法,利用C(2)M的诱饵表达载体筛选果蝇cDNA文库,共发现40个可能与C(2)M相互作用的蛋白,包括多种DNA及组蛋白结合蛋白、ATPase、转录调节因子。从筛选的结果中,选取wech和Psf1基因构建了转基因果蝇,并在生殖细胞中进行了基因沉默,结果显示联会复合体的消失受到延迟。上述结果表明Wech和Psf1蛋白可能与C(2)M形成复合物,共同参与联会复合体的形成或其稳定性的维持。     

Plastid division is mediated by combinatorial assembly of plastid division proteins

Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.     

Interactions between secreted GRA proteins and host cell proteins across the paratitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.     

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

During clathrin‐mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G‐actin) and a central‐acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3‐dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G‐actin‐binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G‐actin‐binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two‐hybrid system, GST‐pulldown, fluorescence polarization and pyrene‐actin polymerization assays, we show that LGM binds G‐actin and is necessary for normal Arp2/3‐mediated actin polymerization in vitro. Live‐cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G‐actin‐binding motif, WH2. These results establish a second G‐actin‐binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.      

TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation

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Signal peptides direct surface proteins to two distinct envelope locations of Staphylococcus aureus

Surface proteins of Gram-positive bacteria are covalently linked to the cell wall envelope by a mechanism requiring an N-terminal signal peptide and a C-terminal LPXTG motif sorting signal. We show here that surface proteins of Staphylococcus aureus arrive at two distinct destinations in the bacterial envelope, either distributed as a ring surrounding each cell or as discrete assembly sites. Proteins with ring-like distribution (clumping factor A (ClfA), Spa, fibronectin-binding protein B (FnbpB), serine-aspartate repeat protein C (SdrC) and SdrD) harbour signal peptides with a YSIRK/GS motif, whereas proteins directed to discrete assembly sites (S. aureus surface protein A (SasA), SasD, SasF and SasK) do not. Reciprocal exchange of signal peptides between surface proteins with (ClfA) or without the YSIRK/GS motif (SasF) directed recombinant products to the alternate destination, whereas mutations that altered only the YSIRK sequence had no effect. Our observations suggest that S. aureus distinguishes between signal peptides to address proteins to either the cell pole (signal peptides without YSIRK/GS) or the cross wall, the peptidoglycan layer that forms during cell division to separate new daughter cells (signal peptides with YISRK/GS motif).     

Characterization of two integral membrane proteins located in the protein bodies of pumpkin seeds

Two integral membrane proteins, MP28 and MP23, were found in protein bodies isolated from pumpkin (Cucurbita sp.) seeds. Molecular characterization revealed that both MP28 and MP23 belong to the seed TIP (tonoplast intrinsic protein) subfamily. The predicted 29 kDa precursor to MP23 includes six putative membrane-spanning domains, and the loop between the first and second transmembrane domains is larger than that of MP28. The N-terminal sequence of the mature MP23 starts from residue 66 in the first loop, indicating that an N-terminal 7 kDa fragment that contains one transmembrane domain is post-translationally removed. During maturation of pumpkin seeds, mRNAs for MP28 and MP23 became detectable in cotyledons at the early stage, and their levels increased slightly until a rapid decrease occurred at the late stage. This is consistent with the accumulation of the 29 kDa precursor and MP28 in the cotyledons at the early stage. By contrast, MP23 appeared at the late stage simultaneously with the disappearance of the 29 kDa precursor. Thus, it seems possible that the conversion of the 29 kDa precursor to the mature MP23 might occur in the vacuoles after the middle stage of seed maturation. Both proteins were localized immunocytochemically on the membranes of the vacuoles at the middle stage and the protein bodies at the late stage. These results suggest that both MP28 and the precursor to MP23 accumulate on vacuolar membranes before the deposition of storage proteins, and then the precursor is converted to the mature MP23 at the late stage. These two TIPs might have a specific function during the maturation of pumpkin seeds.     

Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3-sensitive metalloproteinase

The syndecan family of four transmembrane heparan sulfate proteoglycans binds a variety of soluble and insoluble extracellular effectors. Syndecan extracellular domains (ectodomains) can be shed intact by proteolytic cleavage of their core proteins, yielding soluble proteoglycans that retain the binding properties of their cell surface precursors. Shedding is accelerated by PMA activation of protein kinase C, and by ligand activation of the thrombin (G-protein-coupled) and EGF (protein tyrosine kinase) receptors (Subramanian, S.V., M.L. Fitzgerald, and M. Bernfield. 1997. J. Biol. Chem. 272:14713-14720). Syndecan-1 and -4 ectodomains are found in acute dermal wound fluids, where they regulate growth factor activity (Kato, M., H. Wang, V. Kainulainen, M.L. Fitzgerald, S. Ledbetter, D.M. Ornitz, and M. Bernfield. 1998. Nat. Med. 4:691-697) and proteolytic balance (Kainulainen, V., H. Wang, C. Schick, and M. Bernfield. 1998. J. Biol. Chem. 273:11563-11569). However, little is known about how syndecan ectodomain shedding is regulated.To elucidate the mechanisms that regulate syndecan shedding, we analyzed several features of the process that sheds the syndecan-1 and -4 ectodomains. We find that shedding accelerated by various physiologic agents involves activation of distinct intracellular signaling pathways; and the proteolytic activity responsible for cleavage of syndecan core proteins, which is associated with the cell surface, can act on unstimulated adjacent cells, and is specifically inhibited by TIMP-3, a matrix-associated metalloproteinase inhibitor. In addition, we find that the syndecan-1 core protein is cleaved on the cell surface at a juxtamembrane site; and the proteolytic activity responsible for accelerated shedding differs from that involved in constitutive shedding of the syndecan ectodomains. These results demonstrate the existence of highly regulated mechanisms that can rapidly convert syndecans from cell surface receptors or coreceptors to soluble heparan sulfate proteoglycan effectors. Because the shed ectodomains are found and function in vivo, regulation of syndecan ectodomain shedding by physiological mediators indicates that shedding is a response to specific developmental and pathophysiological cues.