life Review Interplay between Mitochondrial Protein Import and Respiratory Complexes Assembly in Neuronal Health and Degeneration Hope I. Needs 1 , Margherita Protasoni 2, Jeremy M. Henley 1,3, Julien Prudent 2 , Ian Collinson 1,* and Gonçalo C. Pereira 2,* 1 School of Biochemistry, University of Bristol, Bristol BS8 1TD, UK; [email protected] (H.I.N.); [email protected] (J.M.H.) 2 Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, UK; [email protected] (M.P.); [email protected] (J.P.) 3 Centre for Neuroscience and Regenerative Medicine, Faculty of Science, University of Technology Sydney, Ultimo, NSW 2007, Australia * Correspondence: [email protected] (I.C.); [email protected] (G.C.P.) Abstract: The fact that >99% of mitochondrial proteins are encoded by the nuclear genome and synthesised in the cytosol renders the process of mitochondrial protein import fundamental for normal organelle physiology. In addition to this, the nuclear genome comprises most of the proteins required for respiratory complex assembly and function. This means that without fully functional protein import, mitochondrial respiration will be defective, and the major cellular ATP source depleted. When mitochondrial protein import is impaired, a number of stress response pathways are Citation: Needs, H.I.; Protasoni, M.; activated in order to overcome the dysfunction and restore mitochondrial and cellular proteostasis. Henley, J.M.; Prudent, J.; Collinson, I.; However, prolonged impaired mitochondrial protein import and subsequent defective respiratory Pereira, G.C. Interplay between chain function contributes to a number of diseases including primary mitochondrial diseases and Mitochondrial Protein Import and neurodegeneration. This review focuses on how the processes of mitochondrial protein translocation Respiratory Complexes Assembly in and respiratory complex assembly and function are interlinked, how they are regulated, and their Neuronal Health and Degeneration. importance in health and disease. Life 2021, 11, 432. https://doi.org/ 10.3390/life11050432 Keywords: protein import; mitochondrial dysfunction; respiratory complex assembly; supercom- plexes; neurodegeneration; mitochondrial proteostasis Academic Editors: Giorgio Lenaz and Salvatore Nesci Received: 25 March 2021 Accepted: 2 May 2021 1. Introduction Published: 11 May 2021 Mitochondria provide the main source of cellular energy in the form of ATP. This is particularly important in high energy consuming cells such as cardiac and muscle cells as Publisher’s Note: MDPI stays neutral well as in neurons. On top of this vital role in ATP synthesis, mitochondria have a plethora with regard to jurisdictional claims in of other roles, including regulation of cellular metabolism, calcium storage and signalling, published maps and institutional affil- reactive oxygen species (ROS) signalling, damage-associated molecular patterns (DAMPs) iations. production in inflammation and immunity, and programmed cell death [1]. The presence of key enzymes and proteins in different submitochondrial compartments is indispensable for these roles. Consequently, protein translocation becomes a fundamental process for efficient mitochondrial physiology. Due to their diverse proteome, mitochondria have distinct Copyright: © 2021 by the authors. import pathways, which must be fully operational to maintain a healthy organelle [2]. The Licensee MDPI, Basel, Switzerland. first section of this review will cover how cytoplasmic translated proteins are imported This article is an open access article into mitochondria, as well as how mitochondrial-encoded proteins are translocated from distributed under the terms and the matrix to the inner mitochondrial membrane (IMM). We then explore the special conditions of the Creative Commons case of respiratory complexes, which are multimeric proteins assembled from subunits Attribution (CC BY) license (https:// encoded by both the nuclear and mitochondrial genomes to highlight the importance of creativecommons.org/licenses/by/ the import machineries. 4.0/). Life 2021, 11, 432. https://doi.org/10.3390/life11050432 https://www.mdpi.com/journal/life Life 2021, 11, x FOR PEER REVIEW 2 of 46 Life 2021, 11, 432 2 of 44 from subunits encoded by both the nuclear and mitochondrial genomes to highlight the importance of the import machineries. Due to the fundamental importance of mitochondrial homeostasis for the regulation of Due to the fundamental importance of mitochondrial homeostasis for the regulation multiple central processes and pathways, it is not surprising that mitochondrial defects, and of multiple central processes and pathways, it is not surprising that mitochondrial defects, more specifically mitochondrial import defects, have been implicated in several diseases [3]. and more specifically mitochondrial import defects, have been implicated in several dis- These include most neurodegenerative diseases [4], as well as mitochondrial diseases eases [3]. These include most neurodegenerative diseases [4], as well as mitochondrial associated with deficiencies of the respiratory complexes due to mutations affecting the diseases associated with deficiencies of the respiratory complexes due to mutations affect- import machineries [5]. These will be discussed later, followed by a discussion of the recent ing the import machineries [5]. These will be discussed later, followed by a discussion of advancesthe recent therein advances and therein with respect and with to the respect most to common the most mitochondrial common mitochondrial stress response stress and proteostaticresponse and pathways proteostatic thought pathways to counteract thought to import counteract dysfunction. import dysfunction. 2. Protein Translocation 2. Protein Translocation All but 13 of the estimated >1000 human mitochondrial proteins [6] required to All but 13 of the estimated >1000 human mitochondrial proteins [6] required to per- performform key key mitochondrial mitochondrial functions functions are encoded are encoded by the by nucleus the nucleus and synthesised and synthesised on cyto- on cytoplasmicplasmic ribosomes ribosomes and andthus thusmust mustbe imported be imported into mitochond into mitochondriaria through through highly con- highly conservedserved protein protein translocation translocation pathways pathways (Figure (Figure 1). Owing1). Owing to the double to the membrane double membrane bound boundstructure structure of mitochondria, of mitochondria, these multistep these multistep protein translocation protein translocation pathways pathwaysinvolve numer- involve numerousous protein protein complexes complexes (Figure (Figure1 and Table1 and 1). Table Moreover,1). Moreover, their proteome their proteome consists of consists solu- ofble, soluble, membrane membrane-bound,-bound, and transmembrane and transmembrane proteins proteinswith different with differentmitochondrial mitochondrial sub-lo- sub-localisations.calisations. Therefore, Therefore, specialised specialised import machinery import machinery has evolved has to evolved successfully to successfully import importall classes all classes of proteins. of proteins. FigureFigure 1. Overview1. Overview of of human human mitochondrial mitochondrial proteinprotein importimport pathways. The The TOM TOM complex complex acts acts as asthe the central central entry entry gate gate forfor precursor precursor proteins proteins to to enter enter the the IMS, IMS, where where theythey areare diverteddiverted into into one one of of five five pathways, pathways, depending depending onon their their structure, structure, function, and target destination. The MIM pathway (only currently understood in yeast) is an exception in that proteins function, and target destination. The MIM pathway (only currently understood in yeast) is an exception in that proteins usually do not cross the Tom40 channel. Instead, OMM α-helical proteins are recognised by Tom70 and transferred α usuallythrough do notMIM cross to be the inserted Tom40 into channel. the OMM. Instead, The OMMfive major-helical pathways proteins proteins are recognised take after crossing by Tom70 the and TOM transferred channel are through the MIMfollowing. to be inserted The presequence into the OMM. pathway: The five Presequences major pathways containing proteins precursor take after proteins crossing are thetransported TOM channel via the are presequence the following. Thepathway. presequence Of these pathway: proteins, Presequences proteins with containing a hydrophobic precursor sorting proteins sequence are are transported inserted into via the the IMM presequence by the TIM23 pathway.SORT Ofcomplex, these proteins, whereas proteins hydrophilic with amatrix hydrophobic proteins sortingare pulled sequence through are the inserted TIM23MOTOR into complex, the IMM with by the the TIM23 help ofSORT thecomplex, PAM whereascomplex hydrophilic and ATP hydrolysis matrix proteins cycles. are The pulled presequences through of the both TIM23 theseMOTOR groupscomplex, of proteins with are the cleaved help ofby theMPP PAM on the complex matrix and Life 2021, 11, 432 3 of 44 ATP hydrolysis cycles. The presequences of both these groups of proteins are cleaved by MPP on the matrix side. The OXA1 pathway: N-terminally inserted multispanning membrane proteins, once passed through TIM23MOTOR and cleaved by MPP, are passed to OXA1L, which inserts them into the IMM in the N-terminal formation. OXA1L is also responsible