PubMed Health⌕ Search

Biomedical subjects

Nils Johnsson

Publications and source records attributed to Nils Johnsson.

11 recordsLinked to original sources
PubMed2005

Multicolor imaging of cell surface proteins.

We report on a method for the multicolor imaging of cell surface proteins which is based on the labeling of carrier protein (CP) fusion proteins with different fluorophores. In one application, different generations of a cell surface protein can be sequentially labeled to discriminate between old and newly made copies. In another application, fusions to different CPs can be selectively labeled with different fluorophores in one sample. Both applications open up new ways for studying the properties of cell surface proteins of living cells.

Carrier Proteins↗
PubMed2005

Protein kinase CK2 phosphorylates Sec63p to stimulate the assembly of the endoplasmic reticulum protein translocation apparatus.

The heterotetrameric Sec62/63 complex associates with the heterotrimeric Sec61 complex to form the heptameric Sec complex. This complex is necessary and sufficient for post-translational protein translocation across the membrane of the endoplasmic reticulum. We show that Sec63p is phosphorylated at its C-terminal domain by the protein kinase CK2 and that this phosphorylation strengthens the interaction between the cytosolic domains of Sec63p and Sec62p. Exchanging either threonine 652 or threonine 654 against the nonphosphorylatable alanines in Sec63p impairs the binding to Sec62p and interferes with the efficient translocation of proteins across the membrane of the endoplasmic reticulum. These findings show that phosphorylation of Sec63p is required for tightly recruiting the putative signal-sequence-binding subunit Sec62p to the Sec complex.

Casein Kinase II↗
PubMed2005

Protein chemistry on the surface of living cells.

The interplay between carbohydrates, lipids, and proteins determines the stability and flexibility as well as the adhesive and responsive features of the surfaces of all cells. The molecular understanding of the interactions among and between the different classes of these biomolecules is rudimentary at best, a lack of suitable experimental methods being the major reason. Here we discuss a new approach for the specific labeling of fusion proteins of carrier proteins with synthetic compounds on cell surfaces and describe how this approach can be used to investigate the properties of the labeled molecules.

Acyl Carrier Protein↗
PubMed2005

The split-ubiquitin sensor: measuring interactions and conformational alterations of proteins in vivo.

The split-ubiquitin technique can monitor alterations in the conformation of proteins and can be used to detect the interaction between two proteins in living cells. The technique is based on unique features of ubiquitin, the enzymes of the ubiquitin pathway, and the reconstitution of a native-like ubiquitin from its N- and C-terminal fragments. By exploiting the reassociation of a protein from its defined fragments to monitor protein interactions, the split-ubiquitin assay served as the prototype of a still growing number of split-protein sensors to analyze protein function within living cells.

DNA, Single-Stranded↗
PubMed2004

Specific labeling of cell surface proteins with chemically diverse compounds.

The specific and covalent labeling of fusion proteins with synthetic molecules opens up new ways to study protein function in the living cell. Here we present a novel method that allows for the specific and exclusive extracellular labeling of proteins on the surfaces of live cells with a large variety of synthetic molecules including fluorophores, protein ligands, or quantum dots. The approach is based on the specific labeling of fusion proteins of acyl carrier protein with synthetic molecules through post-translational modification catalyzed by phosphopantetheine transferase. The specificity and versatility of the labeling should allow it to become an important tool for studying and manipulating cell surface proteins and for complementing existing approaches in cell surface engineering.

Acyl Carrier Protein↗
PubMed2004

Transforming a (beta/alpha)8--barrel enzyme into a split-protein sensor through directed evolution.

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. We present here a combinatorial method for the generation of new split-protein sensors and demonstrate its application toward the (beta/alpha)(8)-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. This powerful selection complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening.

Aldose-Ketose Isomerases↗
PubMed2003

Pex10p links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome.

The protein import machinery of the peroxisome consists of many proteins, collectively called the peroxins. By applying the split-ubiquitin technique we systematically tested the pair-wise interactions between the Nub- and Cub-labeled peroxins for the first time in the living cells of the yeast Saccharomyces cerevisiae. We found that Pex10p plays a central role in the protein interaction network by connecting the ubiquitin conjugation enzyme Pex4p to the other members of the protein import machinery. A yeast strain harboring a deletion of PEX3 enabled us to estimate the influence of the peroxisomal membrane on the formation of a subset of the investigated protein-protein interactions.

Biological Transport↗
PubMed2003

Covalent and selective immobilization of fusion proteins.

A general method for the covalent immobilization of fusion proteins is presented. The approach is based on the unusual mechanism of the human O6-alkylguanine-DNA alkyltransferase, which irreversibly transfers the alkyl group from its substrate, alkylated or benzylated guanine, to a reactive cysteine residue. By attaching the benzyl group to a surface, hAGT fusion proteins immobilize themselves in a specific and covalent manner. The specificity of the reaction of hAGT with its substrate even allows the specific immobilization of hAGT fusion proteins directly out of cell extracts, making the approach an attractive alternative to currently used immobilization procedures.

Biosensing Techniques↗
PubMed2002

A split-ubiquitin-based assay detects the influence of mutations on the conformational stability of the p53 DNA binding domain in vivo.

Many mutations in the human tumor suppressor p53 affect the function of the protein by destabilizing the structure of its DNA binding domain. To monitor the effects of those mutations in vivo the stability of the DNA binding domain of p53 and some of its mutants was investigated with the split-ubiquitin (split-Ub) method. The split-Ub-derived in vivo data on the relative stability of the mutants roughly correlate with the quantitative data from in vitro denaturation experiments as reported in the literature. A variation of this assay allows visualizing the difference in stability between the wild-type p53 core and the mutant p53(V143A) by a simple growth assay.

Cell Division↗
PubMed2002

Recognition of a subset of signal sequences by Ssh1p, a Sec61p-related protein in the membrane of endoplasmic reticulum of yeast Saccharomyces cerevisiae.

Ssh1p of Saccharomyces cerevisiae is related in sequence to Sec61p, a general receptor for signal sequences and the major subunit of the channel that guides proteins across the membrane of the endoplasmic reticulum. The split-ubiquitin technique was used to determine whether Ssh1p serves as an additional receptor for signal sequences in vivo. We measured the interactions between the N(ub)-labeled Ssh1p and C(ub)-translocation substrates bearing four different signal sequences. The so-determined interaction profile of Ssh1p was compared with the signal sequence interaction profile of the correspondingly modified N(ub)-Sec61p. The assay reveals interactions of Ssh1p with the signal sequences of Kar2p and invertase, whereas Sec61p additionally interacts with the signal sequences of Mfalpha1 and carboxypeptidase Y. The measured physical proximity between Ssh1p and the beta-subunit of the signal sequence recognition particle receptor confirms our hypothesis that Ssh1p is directly involved in the cotranslational translocation of proteins across the membrane of the endoplasmic reticulum.

Endoplasmic Reticulum↗