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PubMed2000

Localization and targeting of SCG10 to the trans-Golgi apparatus and growth cone vesicles.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.

Animals↗
PubMed2000

Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs.

The vesicular stomatitis virus (VSV) G protein is a model transmembrane glycoprotein that has been extensively used to study the exocytotic pathway. A signal in the cytoplasmic tail of VSV G (DxE or Asp-x-Glu, where x is any amino acid) was recently proposed to mediate efficient export of the protein from the endoplasmic reticulum (ER). In this study, we show that the DxE motif only partially accounts for efficient ER exit of VSV G. We have identified a six-amino-acid signal, which includes the previously identified Asp and Glu residues, that is required for efficient exit of VSV G from the ER. This six-residue signal also includes the targeting sequence YxxO (where x is any amino acid and O is a bulky, hydrophobic residue) implicated in several different sorting pathways. The only defect in VSV G proteins with mutations in the six-residue signal is slow exit from the ER; folding and oligomerization in the ER are normal, and the mutants eventually reach the plasma membrane. Addition of this six-residue motif to an inefficiently transported reporter protein is sufficient to confer an enhanced ER export rate. The signal we have identified is highly conserved among divergent VSV G proteins, and we suggest this reflects the importance of this motif in the evolution of VSV G as a proficient exocytic protein.

Amino Acid Sequence↗
PubMed2005

SEC18/NSF-independent, protein-sorting pathway from the yeast cortical ER to the plasma membrane.

Classic studies of temperature-sensitive secretory (sec) mutants have demonstrated that secreted and plasma membrane proteins follow a common SEC pathway via the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles to the cell periphery. The yeast protein Ist2p, which is synthesized from a localized mRNA, travels from the ER to the plasma membrane via a novel route that operates independently of the formation of coat protein complex II-coated vesicles. In this study, we show that the COOH-terminal domain of Ist2p is necessary and sufficient to mediate SEC18-independent sorting when it is positioned at the COOH terminus of different integral membrane proteins and exposed to the cytoplasm. This domain functions as a dominant plasma membrane localization determinant that overrides other protein sorting signals. Based on these observations, we suggest a local synthesis of Ist2p at cortical ER sites, from where the protein is sorted by a novel mechanism to the plasma membrane.

Adenosine Triphosphatases↗
PubMed2004

A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein.

The protozoan parasite Toxoplasma gondii is a highly specialized eukaryote that contains a remarkable number of intracellular compartments, some unique to Apicomplexans and others typical of eukaryotes in general. We have established a green fluorescent protein (GFP)-based motif-trap to identify proteins targeted to different intracellular locations and subsequently the signals responsible for this sorting. The motif-trap involves the transfection and integration of a linearized GFP construct which lacks a promoter and an initiator methionine codon. FACS is used to isolate parasites in which GFP fuses in-frame into a coding region followed by screening by fluorescence microscopy for those containing GFP targeted to specific intracellular compartments. GFP trapping was successful using vectors designed for integration into regions encoding exons and vectors that were engineered with a splice acceptor site for integration into regions encoding introns. This strategy differs from most protein traps in that the resulting fusions are expressed from the endogenous promoter and starting methionine. Thus, problems from inappropriate expression levels or the creation of fortuitous targeting signals seen in library-based traps are diminished. Using this approach, we have trapped GFP localized to a number of intracellular compartments including the nucleus, nucleolus, endoplasmic reticulum, cytosol, parasite surface and rhoptries of Toxoplasma. Further analysis of a parasite clone containing GFP targeted to the rhoptries shows GFP fused to the gene encoding the rhoptry protein ROP4 and has elucidated an additional mechanism for targeting of this protein.

Amino Acid Sequence↗
PubMed2005

Sorting of proteins to storage vacuoles: how many mechanisms?

Vacuoles receive their proteins through the secretory pathway, this requires protein sorting signals and molecular machineries that, until recently, have been believed to be markedly distinct for lytic and storage vacuoles. However, new biochemical, morphological and genetic data indicate that the only known class of vacuolar sorting receptors, believed to be specific for lytic vacuoles, might also be involved in the sorting of certain storage proteins. Furthermore, storage vacuoles can have a complex multimembrane structure that is difficult to explain based on a single trafficking mechanism. A new array of possible molecular interactions is thus emerging that no longer supports a clear-cut distinction between the two types of vacuoles based on sorting signals and putative receptors.

Plant Proteins↗
PubMed2005

Hansenula polymorpha and Saccharomyces cerevisiae Pex5p's recognize different, independent peroxisomal targeting signals in alcohol oxidase.

Peroxisomal alcohol oxidase (AO) from Hansenula polymorpha is inactive and partially mislocalized to the cytosol upon synthesis in Saccharomyces cerevisiae. Co-production with H. polymorpha pyruvate carboxylase (HpPyc1p) resulted in AO activation, but did not improve import into peroxisomes. We show that import of AO mediated by S. cerevisiae Pex5p is strictly dependent on the peroxisomal targeting signal 1 (PTS1) of AO and independent of HpPyc1p. In contrast, HpPex5p-mediated sorting of AO into S. cerevisiae peroxisomes is independent of the PTS1, but requires an alternative PTS that is only formed when HpPyc1p is co-produced and most likely involves folding and co-factor binding to AO.

Alcohol Oxidoreductases↗
PubMed2000

Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5.

Many proteins contain targeting signals within their sequences that specify their delivery to particular organelles. The peroxisomal targeting signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes. The PTS1 sequence closely approximates Ser-Lys-Leu-COO-. PEX5, the receptor for PTS1, interacts with the signal via a series of tetratricopeptide repeats (TPRs) within its C-terminal half. Here we report the crystal structure of a fragment of human PEX5 that includes all seven predicted TPR motifs in complex with a pentapeptide containing a PTS1 sequence. Two clusters of three TPRs almost completely surround the peptide, while a hinge region, previously identified as TPR4, forms a distinct structure that enables the two sets of TPRs to form a single binding site. This structure reveals the molecular basis for PTS1 recognition and demonstrates a novel mode of TPR-peptide interaction.

Amino Acid Motifs↗
PubMed2006

Insulin-dependent phosphorylation of DPP IV in liver. Evidence for a role of compartmentalized c-Src.

Dipeptidyl peptidase IV (DPP IV, CD26, EC 3.4.14.5) serves as a model aimed at elucidating protein sorting signals. We identify here, by MS, several tyrosine-phosphorylated proteins in a rat liver Golgi/endosome (G/E) fraction including DPP IV. We show that a pool of DPP IV is tyrosine-phosphorylated. Maximal phosphorylation was observed after 2 min following intravenous insulin injection. DPP IV coimmunoprecipitated with the cellular tyrosine kinase Src (c-Src) with maximal association also observed after 2 min following insulin injection. DPP IV was found phosphorylated after incubation of nonsolubilized G/E membranes with [gamma-32P]ATP. The c-Src inhibitor PP2 inhibited DPP IV phosphorylation. Oriented proteolysis experiments indicate that a large pool of c-Src is protected in G/E fractions. Following injection of the protein-tyrosine phosphatase inhibitor bpV(phen), DPP IV levels markedly decreased by 40% both in plasma membrane and G/E fractions. In the fraction designated Lh, DPP IV levels decreased by 50% 15 min following insulin injection. Therefore, a pool of DPP IV is tyrosine-phosphorylated in an insulin-dependent manner. The results suggest the presence of a yet to be characterized signalling mechanism whereby DPP IV has access to c-Src-containing signalling platforms.

Amino Acid Sequence↗
PubMed2004

Modulation of the Leishmania donovani peroxin 5 quaternary structure by peroxisomal targeting signal 1 ligands.

The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.

Amino Acid Sequence↗
PubMed1995

Identification of three distinct peroxisomal protein import defects in patients with peroxisome biogenesis disorders.

Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, and classical rhizomelic chondrodysplasia punctata are lethal genetic disorders caused by defects in peroxisome biogenesis. We report here a characterization of the peroxisomal matrix protein import capabilities of fibroblasts from 62 of these peroxisome biogenesis disorder patients representing all ten known complementation groups. Using an immunofluorescence microscopy assay, we identified three distinct peroxisomal protein import defects among these patients. Type-1 cells have a specific inability to import proteins containing the PTS1 peroxisomal targeting signal, type-2 cells have a specific defect in import of proteins containing the PTS2 signal, and type-3 cells exhibit a loss of, or reduction in, the import of both PTS1 and PTS2 proteins. Considering that the common cellular phenotype of Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum's disease has been proposed to be a complete defect in peroxisomal matrix protein import, the observation that 85% (40/47) of the type-3 cell lines imported a low but detectable amount of both PTS1 and PTS2 proteins was surprising. Furthermore, different cell lines with the type-3 defect exhibited a broad spectrum of different phenotypes; some showed a complete absence of matrix protein import while others contained 50-100 matrix protein-containing peroxisomes per cell. We also noted certain relationships between the import phenotypes and clinical diagnoses: both type-1 cell lines were from neonatal adrenoleukodystrophy patients, all 13 type-2 cell lines were from classical rhizomelic chondrodysplasia punctata patients, and the type-3 import defect was found in the vast majority of Zellweger syndrome (22/22), neonatal adrenoleukodytrophy (17/19), and infantile Refsum's disease (7/7) patients. Our finding that all type-1 cell lines were from the second complementation group (CG2), all 13 type-2 cell lines were from CG11, and that cells from the eight remaining complementation groups only exhibit the type-3 defect indicates that mutations in particular genes give rise to the different types of peroxisomal protein import defects. This hypothesis is further supported by correlations between certain complementation groups and particular type-3 subphenotypes: all patient cell lines belonging to CG3 and CG10 showed a complete absence of peroxisomal matrix protein import while those from CG6, CG7, and CG8 imported some peroxisomal matrix proteins. However, the fact that cell lines from within particular complementation groups (CG1, CG4) could have different matrix protein import characteristics suggests that allelic heterogeneity also plays an important role in generating different import phenotypes in certain patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenoleukodystrophy↗
PubMed2000

Sorting and targeting of melanosomal membrane proteins: signals, pathways, and mechanisms.

Newly synthesized melanosomal proteins, like many other cellular proteins, traverse through a series of intracellular compartments en route to melanosomes. Entry and exit of proteins through these compartments is orchestrated by cellular sorting machinery that recognize specific sorting signals. Melanosomal membrane proteins begin their intracellular journey upon co-translational importation into the endoplasmic reticulum (ER). The biosynthetic output of tyrosinase, the key melanogenic enzyme, appears to be regulated by quality-control events at the ER, the 'port of entry' to the secretory pathway. Following maturation in the ER and through the Golgi, the sorting of these proteins in the trans-Golgi network for intracellular retention and transport along endosome/lysosome pathway requires cytoplasmically exposed signals. A di-leucine motif, present in the cytoplasmic tails of most melanosomal proteins, and its interaction with adaptor protein (AP) complexes, specifically AP-3, are critical for these events. Defects in sorting signals and the cytosolic components that interact with these signals result in a number of murine coat color phenotypes and cause human pigmentary disorders. Thus, missense or frame-shift mutations that produce truncated tyrosinase lacking the melanosomal sorting signal(s) appear to be responsible for murine platinum coat color phenotypes and a proportion of human oculocutaneous albinism-1; mutations in AP-3 appear to be responsible for the mocha phenotype in mice and Hermansky-Pudlak-like syndrome in man. Additional signals and sorting steps downstream of AP-3 appear to be required for endosomal sorting and targeting proteins to melanosomes. Signals and mechanisms that sequester melanosomal proteins from endosomes/lysosomes are not understood. Potential candidates that mediate such processes include proteins encoded by lyst and pallid genes. The common occurrence of abnormalities in melanosomes in many storage-pool disorders suggests that melanocytes utilize signals, pathways, and mechanisms shared by other proteins and cell types to assemble a number of specialized proteins and produce unique cell-type-specific organelles.

Amino Acid Sequence↗
PubMed2001

Secretory granule biogenesis: rafting to the SNARE.

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.

Cell Membrane↗
PubMed2000

Sorting of yeast membrane proteins into an endosome-to-Golgi pathway involves direct interaction of their cytosolic domains with Vps35p.

Resident late-Golgi membrane proteins in Saccharomyces cerevisiae are selectively retrieved from a prevacuolar-endosomal compartment, a process dependent on aromatic amino acid-based sorting determinants on their cytosolic domains. The formation of retrograde vesicles from the prevacuolar compartment and the selective recruitment of vesicular cargo are thought to be mediated by a peripheral membrane retromer protein complex. We previously described mutations in one of the retromer subunit proteins, Vps35p, which caused cargo-specific defects in retrieval. By genetic and biochemical means we now show that Vps35p directly associates with the cytosolic domains of cargo proteins. Chemical cross-linking, followed by coimmunoprecipitation, demonstrated that Vps35p interacts with the cytosolic domain of A-ALP, a model late-Golgi membrane protein, in a retrieval signal-dependent manner. Furthermore, mutations in the cytosolic domains of A-ALP and another cargo protein, Vps10p, were identified that suppressed cargo-specific mutations in Vps35p but did not suppress the retrieval defects of a vps35 null mutation. Suppression was shown to be due to an improvement in protein sorting at the prevacuolar compartment. These data strongly support a model in which Vps35p acts as a "receptor" protein for recognition of the retrieval signal domains of cargo proteins during their recruitment into retrograde vesicles.

Alkaline Phosphatase↗
PubMed2001

Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells.

To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking signal variants in mammalian cells, flow cytometry to sort cells based on trafficking phenotype, and quantitative trafficking assays to measure the efficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate that small changes in the local sequence context dramatically alter signal strength, generating a broad spectrum of trafficking phenotypes. Finally, using sequences from our screen, we found that the potency of di-lysine, but not di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha-COP.

Amino Acid Sequence↗
PubMed2005

A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal.

Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.

Bacterial Outer Membrane Proteins↗
PubMed2001

NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell.

The NHE6 protein is a unique Na(+)/H(+) exchanger isoform believed to localize in mitochondria. It possesses a hydrophilic N-terminal portion that is rich in positively charged residues and many hydrophobic segments. In the present study, signal sequences in the NHE6 molecule were examined for organelle localization and membrane topogenesis. When the full-length protein was expressed in COS7 cells, it localized in the endoplasmic reticulum and on the cell surface. Furthermore, the protein was fully N-glycosylated. When green fluorescent protein was fused after the second (H2) or third (H3) hydrophobic segment, the fusion proteins were targeted to the endoplasmic reticulum (ER) membrane. The localization pattern was the same as that of fusion proteins in which green fluorescent protein was fused after H2 of NHE1. In an in vitro system, H1 behaved as a signal peptide that directs the translocation of the following polypeptide chain and is then processed off. The next hydrophobic segment (H2) halted translocation and eventually became a transmembrane segment. The N-terminal hydrophobic segment (H1) of NHE1 also behaved as a signal peptide. Cell fractionation studies using antibodies against the 15 C-terminal residues indicated that NHE6 protein localized in the microsomal membranes of rat liver cells. All of the NHE6 molecules in liver tissue possess an endoglycosidase H-resistant sugar chain. These findings indicate that NHE6 protein is targeted to the ER membrane via the N-terminal signal peptide and is sorted to organelle membranes derived from the ER membrane.

Amino Acid Sequence↗
PubMed2002

The novel EPTP repeat defines a superfamily of proteins implicated in epileptic disorders.

Recent studies suggest that mutations in the LGI1/Epitempin gene cause autosomal dominant lateral temporal epilepsy. This gene encodes a protein of unknown function, which we postulate is secreted. The LGI1 protein has leucine-rich repeats in the N-terminal sequence and a tandem repeat (which we named EPTP) in its C-terminal region. A redefinition of the C-terminal repeat and the application of sensitive sequence analysis methods enabled us to define a new superfamily of proteins carrying varying numbers of the novel EPTP repeats in combination with various extracellular domains. Genes encoding proteins of this family are located in genomic regions associated with epilepsy and other neurological disorders.

Amino Acid Sequence↗
PubMed1995

The tobacco homolog of mammalian calreticulin is present in protein complexes in vivo.

The analysis of protein sorting signals responsible for the retention of reticuloplasmins (RPLs), a group of soluble proteins that reside in the lumen of the endoplasmic reticulum (ER), has revealed a structural similarity between mammalian and plant ER retention signals. We present evidence that the corresponding epitope is conserved in a vast family of soluble ER resident proteins. Microsequences of RPL60 and RPL90, two abundant members of this family, show high sequence similarity with mammalian calreticulin and endoplasmin. RPL60/calreticulin cofractionates and costains with the lumenal binding protein (BiP). Both proteins were detected in the nuclear envelope and the ER, and in mitotic cells in association with the spindle apparatus and the phragmoplast. Immunoprecipitation of proteins from in vivo-labeled cells demonstrated that RPL60/calreticulin is associated with other polypeptides in a stress- and ATP-dependent fashion. RPL60/calreticulin transcript levels increased rapidly in abundance during the proliferation of the secretory apparatus and the onset of hydrolase secretion in gibberellic acid-treated barley aleurone cells. This induction profile is identical to that of the well-characterized ER chaperones BiP and endoplasmin. However, expression patterns in response to different stress conditions as well as tissue-specific expression patterns indicate that these genes are differentially regulated and may not act in concert.

Amino Acid Sequence↗