Biomedical subjects

Peter Schu

Publications and source records attributed to Peter Schu.

9 recordsLinked to original sources

PubMed2005

Gamma-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking.

A novel peripheral membrane protein (2c18) that interacts directly with the gamma 'ear' domain of the adaptor protein complex 1 (AP-1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and gamma1-adaptin at the trans-Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane-bound gamma1-adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady-state levels of gamma1-adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose-6-phosphate receptor at the TGN, which itself involves AP-1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR.

Adaptor Protein Complex 1↗

PubMed2004

AP-1 and AP-3 facilitate lysosomal targeting of Batten disease protein CLN3 via its dileucine motif.

CLN3 is a transmembrane protein with a predominant localization in lysosomes in non-neuronal cells but is also found in endosomes and the synaptic region in neuronal cells. Mutations in the CLN3 gene result in juvenile neuronal ceroid lipofuscinosis or Batten disease, which currently is the most common cause of childhood dementia. We have recently reported that the lysosomal targeting of CLN3 is facilitated by two targeting motifs: a dileucine-type motif in a cytoplasmic loop domain and an unusual motif in the carboxyl-terminal cytoplasmic tail comprising a methionine and a glycine separated by nine amino acids (Kyttala, A., Ihrke, G., Vesa, J., Schell, M. J., and Luzio, J. P. (2004) Mol. Biol. Cell 15, 1313-1323). In the present study, we investigated the pathways and mechanisms of CLN3 sorting using biochemical binding assays and immunofluorescence methods. The dileucine motif of CLN3 bound both AP-1 and AP-3 in vitro, and expression of mutated CLN3 in AP-1- or AP-3-deficient mouse fibroblasts showed that both adaptor complexes are required for sequential sorting of CLN3 via this motif. Our data indicate the involvement of complex sorting machinery in the trafficking of CLN3 and emphasize the diversity of parallel and sequential sorting pathways in the trafficking of membrane proteins.

Adaptor Protein Complex 3↗

PubMed2004

Clathrin adaptor epsinR is required for retrograde sorting on early endosomal membranes.

Retrograde transport links early/recycling endosomes to the trans-Golgi network (TGN), thereby connecting the endocytic and the biosynthetic/secretory pathways. To determine how internalized molecules are targeted to the retrograde route, we have interfered with the function of clathrin and that of two proteins that interact with it, AP1 and epsinR. We found that the glycosphingolipid binding bacterial Shiga toxin entered cells efficiently when clathrin expression was inhibited. However, retrograde transport of Shiga toxin to the TGN was strongly inhibited. This allowed us to show that for Shiga toxin, retrograde sorting on early/recycling endosomes depends on clathrin and epsinR, but not AP1. EpsinR was also involved in retrograde transport of two endogenous proteins, TGN38/46 and mannose 6-phosphate receptor. In conclusion, our work reveals the existence of clathrin-independent and -dependent transport steps in the retrograde route, and establishes a function for clathrin and epsinR at the endosome-TGN interface.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

Rabaptin-5alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes.

Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction with the gamma(1)-subunit. The binding site for gamma(1)-adaptin is in the hinge region of rabaptin-5alpha, which is distinct from rab4- and rab5-binding domains. Endogenous or ectopically expressed gamma(1)- adaptin localized to both the trans-Golgi network and endosomes. Co-expressed rabaptin-5alpha and gamma(1)-adaptin, however, co-localized in a rab4-dependent manner on recycling endosomes. Transfection of rabaptin-5alpha caused enlarged endosomes and delayed recycling of transferrin. RNAi of rab4 had an opposing effect on transferrin recycling. Collectively, our data show that rab4-GTP acts as a scaffold for a rabaptin-5alpha- gamma(1)-adaptin complex on recycling endosomes and that interactions between rab4, rabaptin-5alpha and gamma(1)-adaptin regulate membrane recycling.

Adaptor Protein Complex gamma Subunits↗

PubMed2003

Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin.

The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.

ADP-Ribosylation Factors↗

PubMed2003

Characterization of the in vitro retrograde transport of MPR46.

The mannose 6-phosphate receptor MPR46 mediates sorting of lysosomal enzymes and recycles between the trans-Golgi network and endosomes. We characterized the retrograde transport of MPR46 from endosomes to the TGN by an in vitro transport assay using mouse fibroblast cell lines. Sulfation of a modified MPR46 upon entering the TGN is measured. The in vitro retrograde transport is time-, temperature-, ATP- and cytosol-dependent. Transport requires the SNARE proteins Vti1a and Syntaxin 16 and the Rab family member Rab6. The transport is sensitive to GTP gamma S, brefeldin A and independent of TIP47. These data indicate that MPR46 follows an early endosome-to-TGN route. Transport is inhibited by MPR46 tail peptide comprising the acidic cluster-di-leucine sorting motif to which adaptor proteins AP-1 and AP-3 bind. Transport depends on cytosolic AP-3, but not on cytosolic AP-1. Residual membrane-associated AP-1 may have masked a requirement for cytosolic AP-1. The competence of membranes from AP-1-deficient cells for endosome-to-TGN transport in vitro was severely compromised.

Adaptor Protein Complex 1↗

PubMed2002

The polarized epithelia-specific mu 1B-adaptin complements mu 1A-deficiency in fibroblasts.

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The mu 1-adaptin subunit of the complex exists as the ubiquitous mu 1A and the polarized epithelia-specific mu 1B, which are 80% identical. In polarized epithelia, mu 1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed mu 1B in mu 1A-deficient fibroblasts to test for mu 1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that mu 1A and mu 1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.

Adaptor Protein Complex mu Subunits↗

PubMed2002

AP-1A and AP-3A lysosomal sorting functions.

Heterotetrameric adaptor-protein complexes AP-1A and AP-3A mediate protein sorting in post-Golgi vesicular transport. AP-1A and AP-3A have been localized to the trans-Golgi network, indicating a function in protein sorting at this compartment. AP-3A appears to mediate trans-Golgi network-to-lysosome and also endosome-to-lysosome protein sorting. AP-1A is thought to be required for both trans-Golgi network-to-endosome transport and endosome-to-trans-Golgi network transport. However, the recent discovery of a role for monomeric GGA (Golgi localized gamma-ear containing, ARF binding protein) adaptor proteins in trans-Golgi network to endosome protein transport has brought into question the long-discussed trans-Golgi network-to-endosome sorting function of AP-1A. Murine cytomegalovirus gp48 contains an unusual di-leucine-based lysosome sorting signal motif and mediates lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes, preventing exposure of major histocompatibility complex class I at the plasma membrane. We analyzed lysosomal sorting of gp48/major histocompatibility complex class I receptor complexes in cell lines deficient for AP-1A, AP-3A and both, to determine their sorting functions. We find that AP1-A and AP3-A mediate distinct and sequential steps in the lysosomal sorting. Both sorting functions are required to prevent MHC class I exposure at the plasma membrane at steady-state.

Amino Acid Sequence↗

PubMedDate not supplied

Studies on the biosynthesis of striatal-type diterpenoids and the biological activity of herical.

Feeding experiments with specifically 13C-labeled glucose disclosed that the diterpenoid part of the striatals/striatins is formed via the mevalonate pathway, whereas the pentose moiety originates either via glucuronic acid (70%) or the pentose phosphate cycle (30%). Application of radioactively labeled herical to cultures of Hericium ramosum demonstrates the pivotal role of this cyathane-xyloside in striatal biosynthesis. Herical inhibits a large spectrum of fungi and bacteria and shows cytotoxic and hemolytic properties.

Anti-Bacterial Agents↗