Biomedical subjects

Antje Schäfer

Publications and source records attributed to Antje Schäfer.

6 recordsLinked to original sources

PubMed2005

Yeast Pex14p possesses two functionally distinct Pex5p and one Pex7p binding sites.

Current evidence favors a cycling receptor model for the import of peroxisomal matrix proteins. The yeast Pex14 protein together with Pex13p and Pex17p form the docking subcomplex at the peroxisomal membrane and interact in this cycle with both soluble import receptors Pex5p and Pex7p. In a first step of a structure-function analysis of Saccharomyces cerevisiae Pex14p, we mapped its binding sites with both receptors. Using the yeast two-hybrid system and pull-down assays, we showed that Pex5p directly interacts with two separate regions of ScPex14p, amino acid residues 1-58 and 235-308. The latter binding site at the C terminus of ScPex14p overlaps with a binding site of Pex7p at amino acid residues 235-325. The functional assessment of these two binding sites of ScPex14p with the peroxisomal targeting signal receptors indicates that they have distinct roles. Deletion of the N-terminal 58 amino acids caused a partial defect of matrix protein import in pex14delta cells expressing the Pex14-(59-341)-p fragment; however, it did not lead to a pex phenotype. In contrast, truncation of the C-terminal 106 amino acids of ScPex14p completely blocked this process. On the basis of these and other published data, we propose that the C terminus of Pex14p contains the actual docking site and discuss the possibility that the N terminus could be involved in a Pex5p-Pex14p association inside the peroxisomal membrane.

Binding Sites↗

PubMed2005

Yeast genomics in the elucidation of endoplasmic reticulum (ER) quality control and associated protein degradation (ERQD).

The endoplasmic reticulum (ER) is the eukaryotic organelle where most secreted proteins enter the secretory pathway. They enter this organelle in an unfolded state and are folded by a highly active folding machinery to reach their native state. The ER contains an efficient protein quality control system, which recognizes malfolded and orphan proteins and targets them for elimination by a mechanism called ER-associated degradation (ERAD). Both processes are tightly linked, and they will be abbreviated as ERQD (ER quality control and associated degradation). Because ERQD is highly conserved from yeast to man, the easy amenability of yeast to genetic and molecular biological studies combined with the knowledge of its genome and proteome makes it a preferred organism to study such "housekeeping" functions of eukaryotic cells. New genomic and proteomic methods have led to new experimental concepts. Genome-wide screens using genomic deletion libraries led to the identification of genes involved in the processes in question. Using such a genome-wide approach, we devise a sensitive growth test for selection of yeast mutants defective in ERQD. A chimeric protein (CTL*) was generated consisting of the ER luminal, N-glycosylated CPY* protein fused to a transmembrane domain and cytoplasmic 3-isopropylmalate dehydrogenase, the Leu2 protein. In addition, the nonglycosylated ER-membrane-located ERQD substrate Sec61-2p was fused to Leu2p (Sec61-2-L*). Cells carrying a LEU2 deletion can only grow on medium lacking leucine when the chimeric protein CTL* or Sec61-2-L* is not degraded. Thus, only mutant cells defective in an ERQD component can grow. A genome-wide screen can be performed by transforming the CTL* or Sec61-2-L* coding DNA into the approximately 5000 individual deletion mutants of the EUROSCARF yeast library. Examples for new components required for ERQD found by this method are the mannose-6-phosphate receptor domain protein Yos9p and the ubiquitin domain proteins Dsk2p and Rad23p.

Endoplasmic Reticulum↗

PubMed2005

Endoplasmic reticulum-associated protein quality control and degradation: screen for ERAD mutants after ethylmethane sulfonate mutagenesis.

Proteins destined for secretion in eukaryotic cells enter the endoplasmic reticulum (ER) in an unfolded state and are properly folded in this organelle and sent to their final destination. Misfolded or orphan proteins are retained in the ER by a quality control system, retrotranslocated into the cytosol and degraded. Soluble and membrane proteins were found to require a basic machinery for elimination. It is composed of (1) the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, which polyubiquitinate the substrate proteins during retrotranslocation; (2) the trimeric AAA-ATPase complex Cdc48-Ufd1-Npl4p, which liberates the polyubiquitinated proteins from the ER; and (3) the 26S proteasome, finally degrading the misfolded proteins. Additional components for degradation of soluble or membrane proteins may vary depending on the nature of malfolded proteins. It is therefore of utmost importance to gain insight into the different components of the ER protein quality control and degradation system required for the elimination of the substrate variety. Protein quality control of the ER and subsequent degradation are evolutionarily highly conserved from yeast to human. The yeast Saccharomyces cerevisiae is therefore an elegant model organism for a search of new components of the ER quality control and degradation machinery, because it is easily amenable to genetic and molecular biological experimentation. In this chapter, a genetic approach is presented, which leads to the isolation of mutants and to the identification of proteins involved in protein quality control and ER-associated degradation (ERAD). The method resides in ethylmethane sulfonate (EMS) mutagenesis of a yeast strain followed by screening for stabilization of soluble ERAD substrates, two mutated and consequently malfolded vacuolar enzymes, carboxypeptidase yscY (CPY*) and proteinase yscA (PrA*). Both malfolded proteins are retained in the ER lumen and become substrates of the ERAD machinery.

Endoplasmic Reticulum↗

PubMed2005

Endoplasmic reticulum-associated protein quality control and degradation: genome-wide screen for ERAD components.

In this chapter, a genetic approach is presented that leads to the isolation of mutants and to the identification of proteins involved in protein quality control and endoplasmic reticulum-associated degradation (ERAD). The method makes use of a genomic screen of a yeast deletion library (EUROSCARF). Transformation of each of the approx 5000 strains deleted in one nonvital gene each with a CPY* chimera containing CPY* C-terminally fused to a transmembrane domain and the cytosolic Leu2 protein (3-isopropylmalate dehydrogenase) constitutes the basic screening procedure. Because of a Leu2p deficiency in all deletion strains, cells can grow only when the CTL* chimera is present. As the CPY* module of CTL* will be recognized in ERAD-proficient cells, CTL* will be degraded and the strain is unable to grow. Therefore the absence of genes necessary for ER quality control and ERAD will allow cell growth and indicate the necessity of the respective gene for these processes.

3-Isopropylmalate Dehydrogenase↗

PubMed2004

Functional similarity between the peroxisomal PTS2 receptor binding protein Pex18p and the N-terminal half of the PTS1 receptor Pex5p.

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.

Acyl-CoA Oxidase↗

PubMed2003

Pex15p of Saccharomyces cerevisiae provides a molecular basis for recruitment of the AAA peroxin Pex6p to peroxisomal membranes.

The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.

ATPases Associated with Diverse Cellular Activitie↗