Frank E. Nargang

Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

SUMMARY We present an analysis of over 1,100 of the ∼10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

The Preprotein Translocation Channel of the Outer Membrane of Mitochondria

The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross‐linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.

The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes

The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane β-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.

Kinesin is essential for cell morphogenesis and polarized secretion in Neurospora crassa

Kinesin is a force‐generating molecule that is thought to translocate organelles along microtubules, but its precise cellular function is still unclear. To determine the role of kinesin in vivo, we have generated a kinesin‐deficient strain in the simple cell system Neurospora crassa. Null cells exhibit severe alterations in cell morphogenesis, notably hyphal extension, morphology and branching. Surprisingly, the movement of organelles visualized by video microscopy is hardly affected, but apical hyphae fail to establish a Spitzenkörper, an assemblage of secretory vesicles intimately linked to cell elongation and morphogenesis in Neurospora and other filamentous fungi. As cell morphogenesis depends on polarized secretion, our findings demonstrate that a step in the secretory pathway leading to cell shape determination and cell elongation cannot tolerate a loss of kinesin function. The defect is suggested to affect the transport of small, secretory vesicles to the site involved in protrusive activity, resulting in the uncoordinated insertion of new cell wall material over much of the cell surface. These observations have implications for the presumptive function of kinesin in more complex cell systems.

MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19.

Recognition of targeting signals is a crucial step in protein sorting within the cell. So far, only a few components capable of deciphering targeting signals have been identified, and insights into the chemical nature of the interaction between the signals and their receptors are scarce. Using highly purified mitochondrial outer membrane vesicles, we demonstrate that MOM22 and MOM19, components of the protein import complex of the outer membrane, bind preproteins at the mitochondrial surface in a reversible fashion. Interaction specifically and directly occurs with the N‐terminal presequence and is abolished after inactivation of either MOM22 or MOM19. Binding is salt sensitive, suggesting that recognition involves electrostatic forces between the positive charges of the presequence and the acidic cytosolic domain of MOM22. MOM19 and MOM22 can be cross‐linked with high efficiency. We propose that the two proteins form a complex which functions as the presequence receptor at the mitochondrial surface and facilitates the movement of preproteins into the translocation pore.

Nucleotide sequence of the ribulosebisphosphate carboxylase gene from Rhodospirillum rubrum

SummaryThe nucleotide sequence of a cloned DNA fragment encoding ribulose bisphosphate carboxylase from the purple non-sulfur bacterium Rhodospirillum rubrum has been determined. The deduced amino acid sequence of a 1398 nucleotide open reading frame exhibits weak overall homology to the sequences reported for analogous enzymes from cyanobacteria, algae and angiosperms. Thus knowledge of the sequence is useful in attempts to identify structural features of the enzyme which are essential to catalysis. The gene is flanked by nucleotide sequences similar to those implicated in the initiation of translation and termination of transcription in other bacteria.

Import of cytochrome c heme lyase into mitochondria: a novel pathway into the intermembrane space.

Cytochrome c heme lyase (CCHL) catalyses the covalent attachment of the heme group to apocytochrome c during its import into mitochondria. The enzyme is membrane‐associated and is located within the intermembrane space. The precursor of CCHL synthesized in vitro was efficiently translocated into isolated mitochondria from Neurospora crassa. The imported CCHL, like the native protein, was correctly localized to the intermembrane space, where it was membrane‐bound. As with the majority of mitochondrial precursor proteins, CCHL uses the MOM19‐GIP receptor complex in the outer membrane for import. In contrast to proteins taking the general import route, CCHL was imported independently of both ATP‐hydrolysis and an electrochemical potential as external energy sources. CCHL which lacks a cleavable signal sequence apparently does not traverse the inner membrane to reach the intermembrane space; rather, it translocates through the outer membrane only. Thus, CCHL represents an example of a novel, ‘non‐conservative’ import pathway into the intermembrane space, thereby also showing that the import apparatus in the outer membrane acts separately from the import machinery in the inner membrane.

The Oxa1 Protein Forms a Homooligomeric Complex and Is an Essential Part of the Mitochondrial Export Translocase inNeurospora crassa

The Oxa1 protein is a ubiquitous constituent of the inner membrane of mitochondria. Oxa1 was identified in yeast as a crucial component of the protein export machinery known as the OXA translocase, which facilitates the integration of proteins from the mitochondrial matrix into the inner membrane. We have identified theNeurospora crassa Oxa1 protein which shows a sequence identity of 22% to the yeast homologue. Despite the low level of identity, the function of the homologues is conserved as the N. crassa gene fully complemented a yeast null mutant. Genetic analysis revealed that Oxa1 is essential for viability in N. crassa. Cells propagated under conditions that severely reduce Oxa1 levels grew extremely slowly and were deficient in subunits of complex I and complex IV. Isolation of the Oxa1 complex from N. crassa mitochondria revealed a 170–180-kDa complex that contained exclusively Oxa1. Since the Oxa1 monomer has a molecular weight of 43,000, our data suggest that the OXA translocase consists of a homooligomer most likely containing four Oxa1 subunits.

The Isolated Complex of the Translocase of the Outer Membrane of Mitochondria CHARACTERIZATION OF THE CATION-SELECTIVE AND VOLTAGE-GATED PREPROTEIN-CONDUCTING PORE

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.