Lena Burri

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram‐negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N‐terminal, periplasmic domain rich in POTRA repeats and a C‐terminal beta‐barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two‐partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.

A complete set of SNAREs in yeast.

Trafficking of cargo molecules through the secretory pathway relies on packaging and delivery of membrane vesicles. These vesicles, laden with cargo, carry integral membrane proteins that can determine with which target membrane the vesicle might productively fuse. The membrane fusion process is highly conserved in all eukaryotes and the central components driving membrane fusion events involved in vesicle delivery to target membranes are a set of integral membrane proteins called SNAREs. The yeast Saccharomyces cerevisiae has served as an extremely useful model for characterizing components of membrane fusion through genetics, biochemistry and bioinformatics, and it is now likely that the complete set of SNAREs is at hand. Here, we present the details from the searches for SNAREs, summarize the domain structures of the complete set, review what is known about localization of SNAREs to discrete membranes, and highlight some of the surprises that have come from the search.

Microsporidian mitosomes retain elements of the general mitochondrial targeting system.

Microsporidia are intracellular parasites that infect a variety of animals, including humans. As highly specialized parasites, they are characterized by a number of unusual adaptations, many of which are manifested as extreme reduction at the molecular, biochemical, and cellular levels. One interesting aspect of reduction is the mitochondrion. Microsporidia were long considered to be amitochondriate, but recently a tiny mitochondrion-derived organelle called the mitosome was detected. The molecular function of this organelle remains poorly understood. The mitosome has no genome, so it must import all its proteins from the cytosol. In other fungi, the mitochondrial protein import machinery consists of a network series of heterooligomeric translocases and peptidases, but in microsporidia, only a few subunits of some of these complexes have been identified to date. Here, we look at targeting sequences of the microsporidian mitosomal import system and show that mitosomes do in some cases still use N-terminal and internal targeting sequences that are recognizable by import systems of mitochondria in yeast. Furthermore, we have examined the function of the inner membrane peptidase processing enzyme and demonstrate that mitosomal substrates of this enzyme are processed to mature proteins in one species with a simplified processing complex, Antonospora locustae. However, in Encephalitozoon cuniculi, the processing complex is lost altogether, and the preprotein substrate functions with the targeting leader still attached. This report provides direct evidence for presequencing processing in mitosomes and also shows how a complex molecular system has continued to degenerate throughout the evolution of microsporidia.

A SNARE required for retrograde transport to the endoplasmic reticulum.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are central components of the machinery mediating membrane fusion in all eukaryotic cells. Sequence analysis of the yeast genome revealed a previously uncharacterized SNARE, SNARE-like tail-anchored protein 1 (Slt1). Slt1 is an essential protein localized in the endoplasmic reticulum (ER). It forms a SNARE complex with Sec22 and the ER syntaxin Ufe1. Down-regulation of Slt1 levels leads to improper secretion of proteins normally resident in the ER. We suggest that Slt1 is a component of the SNAREpin required for retrograde traffic to the ER. Based on the previously reported association with Ufe1 and Sec22, Sec20 likely contributes the fourth SNARE to the SNAREpin.

Zim17, a Novel Zinc Finger Protein Essential for Protein Import into Mitochondria

Translocation of precursor proteins across the mitochondrial membranes requires the coordinated action of multisubunit translocases in the outer and inner membrane, and the driving force for translocation across the inner membrane is provided by the matrix-located heat shock protein 70 (mtHsp70). The central components of the protein import machinery are essential. Here we describe Zim17, an essential protein with a zinc finger motif involved in protein import into mitochondria. Comparative genomics suggested a correction to the open reading frame of YNL310c, the gene encoding Zim17 in Saccharomyces cerevisiae. The revised open reading frame codes for a classic mitochondrial targeting signal, which is processed from Zim17 in the mitochondrial matrix. Loss of Zim17 selectively diminishes import of proteins into the matrix of mitochondria, but this loss of Zim17 is partially suppressed by overexpression of the J-protein Pam18/Tim14. We propose that Zim17 functions as an example of a “fractured” J-protein, where a protein like Zim17 contributes a zinc finger domain to Type III J-proteins, in toto providing for substrate loading onto Hsp70.

Integral membrane proteins in the mitochondrial outer membrane of Saccharomyces cerevisiae.

Mitochondria evolved from a bacterial endosymbiont ancestor in which the integral outer membrane proteins would have been β‐barrel structured within the plane of the membrane. Initial proteomics on the outer membrane from yeast mitochondria suggest that while most of the protein components are integral in the membrane, most of these mitochondrial proteins behave as if they have α‐helical transmembrane domains, rather than β‐barrels. These proteins are usually predicted to have a single α‐helical transmembrane segment at either the N‐ or C‐terminus, however, more complex topologies are also seen. We purified the novel outer membrane protein Om14 and show it is encoded in the gene YBR230c. Protein sequencing revealed an intron is spliced from the transcript, and both transcription from the YBR230c gene and steady‐state level of the Om14 protein is dramatically less in cells grown on glucose than in cells grown on nonfermentable carbon sources. Hydropathy predictions together with data from limited protease digestion show three α‐helical transmembrane segments in Om14. The α‐helical outer membrane proteins provide functions derived after the endosymbiotic event, and require the translocase in the outer mitochondrial membrane complex for insertion into the outer membrane.

A Broad Distribution of the Alternative Oxidase in Microsporidian Parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.