title: In vitro and in vivo approaches to studying the bacterial signal peptide processing.
abstract: Protein targeting in both eukaryotic and prokaryotic cells is often directed by a signal sequence located at the amino-terminus of the protein. In eukaryotes, proteins that are sorted into different compartments of the cell, such as endoplasmic reticulum, mitochondria, and chloroplast, require different signal sequences. In bacteria, proteins which are exported to the outer membrane or the periplasmic space are also guided by signal peptides. After the protein is translocated across the cytoplasmic membrane, the signal peptide is proteolytically removed by signal peptide cleavage. Here, in this chapter, we describe methods to study signal peptide processing in bacteria, including purification of signal peptidase and its substrates. We also describe the measurement of the catalytic constants of signal peptidases using an in vitro assay. In addition, we will present an in vivo assay using a temperature sensitive signal peptidase strain to determine which preproteins are processed by Signal peptidase 1.
authors.affiliation: Department of Chemistry, The Ohio State University, Columbus, OH, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Wang
authors.forename: Ross E
authors.initials: RE
authors.lastname: Dalbey
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology; D012697:Serine Endopeptidases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D012697:Serine Endopeptidases; C026515:type I signal peptidase