Applications of peptide arrays prepared by the SPOT-technology

Reineke, Ulrich; Volkmer, Rudolf; Schneider‐Mergener, Jens

doi:10.1016/s0958-1669(00)00178-6

Cited by 241 publications

(130 citation statements)

References 47 publications

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“…The peptide arrays were synthesized on modified cellulose membranes termed CAPE membranes (Landgraf et al, 2004) using the SPOT synthesis technique (Reineke et al, 2001). In vitro binding of Pex19p to the peptide libraries was analyzed as follows: the soluble fraction of glutathione Stransferase (GST)-Pex19p that had been expressed in C41 (DE3) from plasmid pKat79 (PEX19 in pGEX4T-2) was bound for 2 h at 4°C on a glutathioneSepharose 4B matrix (Amersham Pharmacia).…”

Section: Peptide Blot Assaysmentioning

confidence: 99%

Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

Rottensteiner

Kramer

Lorenzen

et al. 2004

MBoC

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162

196

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Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane. As a consequence, targeting signals of PMPs (mPTS) are rather complex. A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs. However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS. By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae. The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids. With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix. A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site. By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored. We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS. INTRODUCTIONPeroxisomes are ubiquitous organelles of eukaryotic cells, whose proteins are imported posttranslationally. Matrix proteins are directed to peroxisomes by either of two targeting signals, a C-terminal PTS1 or an N-terminal PTS2. The topogenesis of peroxisomal membrane proteins (PMPs) is accomplished by yet another mechanism, because most of the peroxin mutants, characterized by their defect in the biogenesis of peroxisomes, exhibit a block in matrix protein import, but do import PMPs normally (Lazarow and Fujiki, 1985;Gould and Valle, 2000;Subramani et al., 2000;Purdue and Lazarow, 2001;Eckert and Erdmann, 2003). To date, only three peroxins with a potential role in PMP targeting have been identified, namely Pex3p (Hettema et al., 2000;South et al., 2000), Pex16p in mammals (South and Gould, 1999;Honsho et al., 2002), and Pex19p (Gö tte et al., 1998;Matsuzono et al., 1999;Snyder et al., 1999;Soukupova et al., 1999). In cells lacking any of these proteins, PMPs are either degraded or mistargeted to other subcellular compartments such as mitochondria, the endoplasmic reticulum (ER) and membranes of unknown origin (Ghaedi et al., 2000;Hettema et al., 2000;Sacksteder et al., 2000).In accordance with a distinct pathway, PMPs use neither PTS1 nor PTS2. The targeting signals of PMPs (mPTS) that direct and insert PMPs into the peroxisomal membrane have been determined for a number of PMPs of several species. Despite some differences, a picture emerged from these studies of a targeting signal consisting of one or more transmembrane domains in conjunction with a short sequence, which contains either a cluster of basic residues or a mixture of basic and hydrophobic amino acids (Dyer et al., 1996;Baerends et al., 2000b;Pause et al., 2000;...

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Section: Peptide Blot Assaysmentioning

confidence: 99%

Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

Rottensteiner

Kramer

Lorenzen

et al. 2004

MBoC

Self Cite

162

196

View full text Add to dashboard Cite

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“…Arrays of tissues, peptides, antibodies, proteins, and even cells have been developed (MacBeath and Schreiber, 2000;Haab et al, 2001;Reineke et al, 2001;Ziauddin and Sabatini, 2001). This is further evidence of the strength and versatility for high throughput screening.…”

Section: Molecular Studies Using Microarray Platforms For Target Discmentioning

confidence: 99%

Applications of array technology: identification of molecular targets in bladder cancer

Sänchez‐Carbayo

Cordon‐Cardo

2003

Br J Cancer

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“…So far, direct binding of N-Sst2 to Vps36 and Tlg2 has been demonstrated using synthetic peptide arrays (80). 3 This analysis revealed binding to three discontinuous peptide "epitopes" within Vps36 and a single segment within Tlg2.…”

Section: Discussionmentioning

confidence: 95%

Regulation of Stress Response Signaling by the N-terminal Dishevelled/EGL-10/Pleckstrin Domain of Sst2, a Regulator of G Protein Signaling in Saccharomyces cerevisiae

Burchett¹,

Flanary²,

Aston³

et al. 2002

Journal of Biological Chemistry

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All members of the regulator of G protein signaling (RGS) family contain a conserved core domain that can accelerate G protein GTPase activity. The RGS in yeast, Sst2, can inhibit a G protein signal leading to mating. In addition, some RGS proteins contain an N-terminal domain of unknown function. Here we use complementary whole genome analysis methods to investigate the function of the N-terminal Sst2 domain. To identify a signaling pathway regulated by N-Sst2, we performed genomewide transcription profiling of cells expressing this fragment alone and found differences in 53 transcripts. Of these, 40 are induced by N-Sst2, and nearly all contain a stress response element (STRE) in the promoter region. To identify components of a signaling pathway leading from N-Sst2 to STREs, we performed a genomewide two-hybrid analysis using N-Sst2 as bait and found 17 interacting proteins. To identify the functionally relevant interacting proteins, we analyzed all of the available gene deletion mutants and found three (vps36⌬, pep12⌬, and tlg2⌬) that induce STRE and also repress pheromone-dependent transcription. We selected VPS36 for further characterization. A vps36⌬ mutation diminishes signaling by pheromone as well as by downstream components including the G protein, effector kinase (Ste11), and transcription factor (Ste12). Conversely, overexpression of Vps36 enhances the pheromone response in sst2⌬ cells but not in wild type. These findings indicate that Vps36 and Sst2 have opposite and opposing effects on the pheromone and stress response pathways, with Vps36 acting downstream of the G protein and independently of Sst2 RGS activity.

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Applications of peptide arrays prepared by the SPOT-technology

Cited by 241 publications

References 47 publications

Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

Applications of array technology: identification of molecular targets in bladder cancer

Regulation of Stress Response Signaling by the N-terminal Dishevelled/EGL-10/Pleckstrin Domain of Sst2, a Regulator of G Protein Signaling in Saccharomyces cerevisiae

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