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

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.

Biogenesis of mitochondrial β-barrel membrane proteins.

β-barrel membrane proteins in the mitochondrial outer membrane are crucial for mediating the metabolite exchange between the cytosol and the mitochondrial intermembrane space. In addition, the β-barrel membrane protein subunit Tom40 of the translocase of the outer membrane (TOM) is essential for the import of the vast majority of mitochondrial proteins encoded in the nucleus. The sorting and assembly machinery (SAM) in the outer membrane is required for the membrane insertion of mitochondrial β-barrel proteins.

Role of the small protein Mco6 in the mitochondrial sorting and assembly machinery.

The majority of mitochondrial precursor proteins are imported through the Tom40 β-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for β-barrel membrane protein insertion into the outer membrane and thus required for the assembly of the TOM complex. Here, we demonstrate that the α-helical outer membrane protein Mco6 co-assembles with the mitochondrial distribution and morphology protein Mdm10 as part of the SAM machinery.

Central role of Tim17 in mitochondrial presequence protein translocation.

The presequence translocase of the mitochondrial inner membrane (TIM23) represents the major route for the import of nuclear-encoded proteins into mitochondria. About 60% of more than 1,000 different mitochondrial proteins are synthesized with amino-terminal targeting signals, termed presequences, which form positively charged amphiphilic α-helices. TIM23 sorts the presequence proteins into the inner membrane or matrix.

A multipoint guidance mechanism for β-barrel folding on the SAM complex.

Mitochondrial β-barrel proteins are essential for the transport of metabolites, ions and proteins. The sorting and assembly machinery (SAM) mediates their folding and membrane insertion. We report the cryo-electron microscopy structure of the yeast SAM complex carrying an early eukaryotic β-barrel folding intermediate.

Widespread polycistronic gene expression in green algae.

Polycistronic gene expression, common in prokaryotes, was thought to be extremely rare in eukaryotes. The development of long-read sequencing of full-length transcript isomers (Iso-Seq) has facilitated a reexamination of that dogma. Using Iso-Seq, we discovered hundreds of examples of polycistronic expression of nuclear genes in two divergent species of green algae: and Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci.

Methods for studying protein targeting to and within the chloroplast.

Distinct protein complements impart each of the chloroplast's three membranes and three aqueous spaces with specific functions essential for plant growth and development. Chloroplasts capture light energy, synthesize macromolecular building blocks and specialized metabolites, and communicate environmental signals to the nucleus. Establishing and maintaining these processes requires approximately 3000 proteins derived from nuclear genes, constituting approximately 95% of the chloroplast proteome.

Structural considerations of folded protein import through the chloroplast TOC/TIC translocons.

Protein import into chloroplasts is carried out by the protein translocons at the outer and inner envelope membranes (TOC and TIC). Detailed structures for these translocons are lacking, with only a low-resolution TOC complex structure available. Recently, we showed that the TOC/TIC translocons can import folded proteins, a rather unique feat for a coupled double membrane system.

Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays.

Chloroplasts are the organelles in green plants responsible for carrying out numerous essential metabolic pathways, most notably photosynthesis. Within the chloroplasts, the thylakoid membrane system houses all the photosynthetic pigments, reaction center complexes, and most of the electron carriers, and is responsible for light-dependent ATP synthesis. Over 90% of chloroplast proteins are encoded in the nucleus, translated in the cytosol, and subsequently imported into the chloroplast.

Evaluating the Functional Pore Size of Chloroplast TOC and TIC Protein Translocons: Import of Folded Proteins.

The degree of residual structure retained by proteins while passing through biological membranes is a fundamental mechanistic question of protein translocation. Proteins are generally thought to be unfolded while transported through canonical proteinaceous translocons, including the translocons of the outer and inner chloroplast envelope membranes (TOC and TIC). Here, we readdressed the issue and found that the TOC/TIC translocons accommodated the tightly folded dihydrofolate reductase (DHFR) protein in complex with its stabilizing ligand, methotrexate (MTX).