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

The majority of mitochondrial precursor proteins are imported through the Tom40 ß-barrel channel of the translocase of the outer membrane (TOM). The sorting and assembly machinery (SAM) is essential for ß-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. MCO6 and MDM10 display a negative genetic interaction, and a mco6-mdm10 yeast double mutant displays reduced levels of the TOM complex. Cells lacking Mco6 affect the levels of Mdm10 and show assembly defects of the TOM complex. Thus, this work uncovers a role of the SAMMco6 complex for the biogenesis of the mitochondrial outer membrane.

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 mitochondria1,2. 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-helices3,4. TIM23 sorts the presequence proteins into the inner membrane or matrix. Various views, including regulatory and coupling functions, have been reported on the essential TIM23 subunit Tim17 (refs. 5-7). Here we mapped the interaction of Tim17 with matrix-targeted and inner membrane-sorted preproteins during translocation in the native membrane environment. We show that Tim17 contains conserved negative charges close to the intermembrane space side of the bilayer, which are essential to initiate presequence protein translocation along a distinct transmembrane cavity of Tim17 for both classes of preproteins. The amphiphilic character of mitochondrial presequences directly matches this Tim17-dependent translocation mechanism. This mechanism permits direct lateral release of transmembrane segments of inner membrane-sorted precursors into the inner membrane.

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

Mitochondrial ß-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 ß-barrel folding intermediate. The lateral gate of Sam50 is wide open and pairs with the last ß-strand (ß-signal) of the substrate-the 19-ß-stranded Tom40 precursor-to form a hybrid barrel in the membrane plane. The Tom40 barrel grows and curves, guided by an extended bridge with Sam50. Tom40's first ß-segment (ß1) penetrates into the nascent barrel, interacting with its inner wall. The Tom40 amino-terminal segment then displaces ß1 to promote its pairing with Tom40's last ß-strand to complete barrel formation with the assistance of Sam37's dynamic α-protrusion. Our study thus reveals a multipoint guidance mechanism for mitochondrial ß-barrel folding.

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: Chlamydomonas reinhardtii and Chromochloris zofingiensis Here, we employ a range of independent approaches to validate that multiple proteins are translated from a common transcript for hundreds of loci. A chromatin immunoprecipitation analysis using trimethylation of lysine 4 on histone H3 marks confirmed that transcription begins exclusively at the upstream gene. Quantification of polyadenylated [poly(A)] tails and poly(A) signal sequences confirmed that transcription ends exclusively after the downstream gene. Coexpression analysis found nearly perfect correlation for open reading frames (ORFs) within polycistronic loci, consistent with expression in a shared transcript. For many polycistronic loci, terminal peptides from both ORFs were identified from proteomics datasets, consistent with independent translation. Synthetic polycistronic gene pairs were transcribed and translated in vitro to recapitulate the production of two distinct proteins from a common transcript. The relative abundance of these two proteins can be modified by altering the Kozak-like sequence of the upstream gene. Replacement of the ORFs with selectable markers or reporters allows production of such heterologous proteins, speaking to utility in synthetic biology approaches. Conservation of a significant number of polycistronic gene pairs between C. reinhardtii, C. zofingiensis, and five other species suggests that this mechanism may be evolutionarily ancient and biologically important in the green algal lineage.

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. These proteins are imported into chloroplasts from the cytosol, sorted to the correct subcompartment, and assembled into functioning complexes. In vitro import assays can reconstitute these processes in isolated chloroplasts. We describe methods for monitoring in vitro protein import using Pisum sativum chloroplasts and for protease protection, fractionation, and native protein electrophoresis that are commonly combined with the import assay. These techniques facilitate investigation of the import and sorting processes, of where a protein resides, and of how that protein functions.

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. We also determined the maximum functional TOC/TIC pore size to be 30-35 Å. Here, we discuss how such large pores could form and compare the structural dynamics of the pore-forming Toc75 subunit to its bacterial/mitochondrial Omp85 family homologs. We put forward structural models that can be empirically tested and also briefly review the pore dynamics of other protein translocons with known structures.

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. Further protein transport into or across the thylakoid membrane utilizes one of four translocation pathways. Here, we describe a high-yield method for isolation of transport-competent thylakoids from peas (Pisum sativum), along with transport assays through the three energy-dependent cpTat, cpSec1, and cpSRP-mediated pathways. These methods enable experiments relating to thylakoid protein localization, transport energetics, and the mechanisms of protein translocation across biological membranes.

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). We employed a fluorescein-conjugated methotrexate (FMTX), which has slow membrane transport rates relative to unconjugated MTX, to show that the rate of ligand accumulation inside chloroplasts is faster when bound to DHFR that is actively being imported. Stromal accumulation of FMTX is ATP dependent when DHFR is actively being imported but is otherwise ATP independent, again indicating DHFR/FMTX complex import. Furthermore, the TOC/TIC pore size was probed with fixed-diameter particles and found to be greater than 25.6 Å, large enough to support folded DHFR import and also larger than mitochondrial and bacterial protein translocons that have a requirement for protein unfolding. This unique pore size and the ability to import folded proteins have critical implications regarding the structure and mechanism of the TOC/TIC translocons.