Review Proteinimportintomitochondriaof Neurospora crassa HolgerProkisch, * StephanNussberger,andBenediktWestermann Institut f€ ur Physiologische Chemie, Universit€ atM€ unchen, Butenandtstr. 5, 81377 Munich, Germany Received8March2002;accepted14March2002 Abstract Biogenesis of mitochondria requires import of several hundreds of different nuclear-encoded preproteins needed for mito- chondrialstructureandfunction.Importandsortingofthesepreproteinsisamultistepprocessfacilitatedbycomplexproteinaceous machinerieslocatedinthemitochondrialouterandinnermembranes.Thetranslocaseofthemitochondrialoutermembrane,the TOM complex, comprises receptors which specifically recognize mitochondrial preproteins and a protein conducting channel formedbyTOM40.TheTOMcomplexisabletoinsertresidentproteinsintotheoutermembraneandtotranslocateproteinsinto theintermembranespace.Forimportofinnermembraneormatrixproteins,theTOMcomplexcooperateswithtranslocasesofthe innermembrane,theTIMcomplexes.Duringthepast30years,intenseresearchonfungienabledtheidentificationandmechanistic characterizationofanumberofdifferentproteinsinvolvedinproteintranslocation.Thisreviewfocusesonthecontributionsofthe filamentousfungus Neurospora crassa toourcurrentunderstandingofmitochondrialproteinimport,withspecialemphasisonthe structureandfunctionoftheTOMcomplex. Ó 2002ElsevierScience(USA).Allrightsreserved. Keywords: Biogenesis;Mitochondria; Neurospora crassa;Proteinimport;TOM;TIM 1. Introduction Mitochondria supply the cell with energy generated by oxidative phosphorylation (Saraste, 1999). Further- more, they are the compartment for many other im- portant metabolic processes, including reactions of the tricarboxylic acid cycle, iron/sulfur cluster assembly, andbiosynthesisofmanycellularmetabolites(Scheffler, 2001).Mitochondriacannotbegenerateddenovo,but grow from preexisting organelles. Despite the capacity ofmitochondriatoencodeandsynthesizepolypeptides, the major part of their proteome is encoded by a vast arrayofgeneslocatedinthenucleus.Infungi,asinmost eukaryoticorganisms,onlyahandfulofgeneswerere- tained in the mitochondrial genome during evolution (Gray et al., 1999). These encode mostly hydrophobic componentsoftherespiratorychainandribosomalsu- bunits. Several hundred mitochondrial proteins are synthesized on cytosolic ribosomes and have to be im- portedintomitochondria.Thisrequirescomplexprotein machineriesfortheimportandsortingofpolypeptides (Neupert,1997). The molecular machinery that mediates protein translocation into mitochondria has been conserved during the evolution of eukaryotic cells. Fungi are ex- cellent model organisms for studying this process be- causegeneticandbiochemicalapproachescanbereadily combined. Distinct advantages of the filamentous fun- gus Neurospora crassa and the budding yeast Saccha- romyces cerevisiae enabled major discoveries of mitochondrial biology. The characterization of the mi- tochondrialproteinimportcomplexesinyeasthasbeen discussedinseveralexcellentrecentreviews(Herrmann and Neupert, 2000; Neupert, 1997; Pfanner and Geiss- ler, 2001). Why has Neurospora proven to be an ex- tremely valuable model organism for the study of mitochondrialbiogenesis?Earlygeneticstudiescertified Neurospora as a genetically tractable organism and demonstratedthatitshaploidconstitution,easyculture conditions,andsusceptibilitytomutagenesismakeitan idealeukaryoticsystemforgeneticstudiesthatallowthe accumulation of a wealth of biological and genetic information (Davis, 2000; Perkins, 1992; Perkins and Davis, 2000). In the past 3 decades especially, the FungalGeneticsandBiology36(2002)85–90 www.academicpress.com * Correspondingauthor.Fax:+49-89-2180-7093. E-mail address: prokisch@lmu.de(H.Prokisch). 1087-1845/02/$-seefrontmatter Ó 2002ElsevierScience(USA).Allrightsreserved. PII:S1087-1845(02)00018-X