Cytochromes of c-type contain covalently attached hemes that are formed via thioether bonds between the vinyls of heme b and cysteines of the C1XXC2H motifs of apocytochromes. In various organisms, this post-translational modification relies on membrane-associated specific biogenesis proteins, referred to as cytochrome c maturation systems. A highly complex version of these systems, Ccm or System I, is found in Gram-negative bacteria, archaea and plant mitochondria. Here, we describe emerging functional interactions between the Ccm components categorized into three conserved modules, and present a mechanistic view of the molecular basis of ubiquitous vinyl-2~Cys1 and vinyl-4~Cys2 heme b-apocytochrome c thioether bonds in c-type cytochromes.

The twin arginine translocation (Tat) system is a machinery which can translocate folded proteins across energy transducing membranes. Currently it is supposed that Tat substrates bind directly to Tat translocon components before a ApH-driven translocation occurs. In this review, an alternative model is presented which proposes that membrane integration could precede Tat-dependent translocation. This idea is mainly supported by the recent observations of Tat-independent membrane insertion of Tat substrates in vivo and in vitro. Membrane insertion may allow i) a quality control of the folded state by membrane bound proteases like FtsH, ii) the recognition of the membrane spanning signal peptide by Tat system components, and iii) a pulling mechanism of translocation. In some cases of folded Tat substrates, the membrane targeting process may require ATP-dependent N-terminal unfolding-steps.

In eukaryotic cells, the reversible attachment of small ubiquitin-like modifier (SUMO) protein is a post-translational modification that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner enhances functional protein production in prokaryotic and eukaryotic expression systems, based upon significantly improved protein stability and solubility. Following the expression and purification of the fusion protein, the SUMO-tag can be cleaved by specific (SUMO) proteases via their endopeptidase activity in vitro to generate the desired N-terminus of the released protein partner. In addition to its physiological relevance in eukaryotes, SUMO can, thus, be used as a powerful biotechnological tool for protein expression in prokaryotic and eukaryotic cell systems.In this chapter, we will describe the construction of a fusion protein with the SUMO-tag, its expression in Escherichia coli, and its purification followed by the removal of the SUMO-tag by a SUMO-specific protease in vitro.