bims-tricox 2024-11-10 papers

Issue of 2024‒11‒10
four papers selected by
Yash Verma, University of Zurich

Methods Enzymol. 2024 ;pii: S0076-6879(24)00402-6. [Epub ahead of print]707 257-298

Analysis of mitochondrial protein translocation by disulfide bond formation and cysteine specific crosslinking.

Laura F Fielden, Jakob D Busch, Caroline Lindau, Jian Qiu, Nils Wiedemann.

Protein translocation is a highly dynamic process and, in addition, mitochondrial protein import is especially complicated as the majority of nuclear encoded precursor proteins must engage with multiple translocases before they are assembled in the correct mitochondrial subcompartment. In this chapter, we describe assays for engineered disulfide bond formation and cysteine specific crosslinking to analyze the rearrangement of translocase subunits or to probe protein-protein interactions between precursor proteins and translocase subunits. Such assays were used to characterize the translocase of the outer membrane, the presequence translocase of the inner membrane and the sorting and assembly machinery for the biogenesis of β-Barrel proteins. Moreover, these approaches were also employed to determine the translocation path of precursor proteins (identification of import receptors and specific domains required for translocation) as well as the analysis, location and translocase subunit dependence for the formation of β-Barrel proteins. Here we describe how engineered disulfide bond formation and cysteine specific crosslinking assays are planned and performed and discuss important aspects for its application to study mitochondrial protein translocation.

Keywords: Mitochondria; Precursor protein; Protein biogenesis; Protein import; Protein-protein interaction; SAM complex; TIM complex; TOM complex; Translocase

DOI: https://doi.org/10.1016/bs.mie.2024.07.057

Methods Enzymol. 2024 ;pii: S0076-6879(24)00400-2. [Epub ahead of print]707 39-62

Monitoring α-helical membrane protein insertion into the outer mitochondrial membrane of yeast cells.

Isabelle Steymans, Thomas Becker.

Mitochondria are surrounded by two membranes, the outer and inner membrane. The outer membrane contains a few dozen integral membrane proteins that mediate transport, fusion and fission processes, form contact sites and are involved in signaling pathways. There are two different types of outer membrane proteins. A few proteins are membrane-integrated by a transmembrane β-barrel, while other proteins are embedded by single or multiple α-helical membrane segments. All outer membrane proteins are produced on cytosolic ribosomes, but their import mechanisms differ. The translocase of the outer mitochondrial membrane (TOM complex) and the sorting and assembly machinery (SAM complex) import β-barrel proteins. Different import pathways have been reported for proteins with α-helical membrane anchors. The mitochondrial import (MIM) complex is the major insertase for this type of proteins. The in vitro import of radiolabeled precursor proteins into isolated mitochondria is a versatile technique to study protein import into the outer mitochondrial membrane. The import of these proteins does not involve proteolytic processing and is not dependent on the membrane potential. Therefore, the import assay has to be combined with blue native electrophoresis, carbonate extraction or protease accessibility assays to determine the import efficiency. These techniques allow to define import steps, assembly intermediates and study membrane integration. The in vitro import assay has been a central tool to uncover specific import routes and mechanisms.

Keywords: MIM complex; Mitochondria; Protein sorting; SAM complex; TOM complex

DOI: https://doi.org/10.1016/bs.mie.2024.07.055

Methods Enzymol. 2024 ;pii: S0076-6879(24)00383-5. [Epub ahead of print]707 367-388

Large-scale purification of mitochondrial protein complexes in yeast expression system for structural analyses.

Nanako Kobayashi, Yuta Imai, Shintaro Kita, Shiho Kawai, Yuhei Araiso.

Recent developments in cryo-electron microscopy techniques have facilitated intensive research into determining protein structures. Nevertheless, the structures of some mitochondrial membrane protein complexes remain undetermined. One possible reason for this research gap is that mitochondrial membrane protein complexes are difficult to overexpress and purify. Even using high-resolution cryo-electron microscopy, structural determination is not possible without first obtaining purified homogeneous proteins. As determining novel structures of protein complexes would provide opportunities to answer many unresolved biological questions, it is important to generalize purification methods, which often become bottlenecks in protein research. In this chapter, we introduce purification methods for mitochondrial membrane protein complexes and mitochondria-localized soluble protein complexes using a yeast expression system. We also describe the recent development of a mitochondrial membrane isolation method that enables the extraction of large amounts of protein complexes for structural analyses.

Keywords: Mitochondria preparation; Mitochondrial proteins; Multi-gene expression; Protein complex purification; Structural analysis; Yeast expression system

DOI: https://doi.org/10.1016/bs.mie.2024.07.043

Methods Enzymol. 2024 ;pii: S0076-6879(24)00380-X. [Epub ahead of print]707 611-634

Screen for temperature-sensitive mutants of non-essential yeast genes.

Iniyan Ganesan, Nikolaus Pfanner, Nils Wiedemann.

Yeast deletion mutants of crucial genes are often associated with a number of secondary defects, which hamper the analysis of primary protein function. Therefore, temperature-sensitive mutants are valuable tools to evaluate protein function in a focused and often reversible manner. However, temperature-sensitive mutants are uncommon for non-essential genes that nevertheless may have strong defects. Here we describe a screening method for generating temperature-sensitive mutants of non-essential genes in synthetic lethal backgrounds of Saccharomyces cerevisiae. As proof of principle, we describe a successful screen for the yeast mitochondrial inner membrane protease iAAA subunit Yme1 utilizing two screening approaches: a random mutagenesis and rational design approach. We then describe how candidate temperature-sensitive mutants are validated.

Keywords: Mitochondria; Protease; Proteolysis; Saccharomyces cerevisiae; Temperature-sensitive screen; Yeast; Yme1; i-AAA

DOI: https://doi.org/10.1016/bs.mie.2024.07.040