The development of intracytoplasmic membranes in alphaproteobacteria involves the conserved mitochondrial crista-developing protein Mic60

Abstract

Mitochondrial cristae expand the surface area of respiratory membranes and ultimately allow for the evolutionary scaling of respiration with cell volume across eukaryotes. The discovery of Mic60 homologs among alphaproteobacteria, the closest extant relatives of mitochondria, suggested that cristae might have evolved from bacterial intracytoplasmic membranes (ICMs). Here, we investigated the predicted structure and function of alphaproteobacterial Mic60, and a protein encoded by an adjacent gene Orf52, in two distantly related purple alphaproteobacteria, Rhodobacter sphaeroides and Rhodopseudomonas palustris. In addition, we assessed the potential physical interactors of Mic60 and Orf52 in R. sphaeroides. We show that the three alpha-helices of mitochondrial Mic60's mitofilin domain, as well as its adjacent membrane-binding amphipathic helix, are present in alphaproteobacterial Mic60. The disruption of Mic60 and Orf52 caused photoheterotrophic growth defects, which are most severe under low light conditions, and both their disruption and overexpression lead to enlarged ICMs in both studied alphaproteobacteria. We also found that alphaproteobacterial Mic60 physically interacts with BamA, the homolog of Sam50, one of the main physical interactors of eukaryotic Mic60. This interaction, responsible for making contact sites at mitochondrial envelopes, has been conserved in modern alphaproteobacteria despite more than a billion years of evolutionary divergence. Our results suggest a role for Mic60 in photosynthetic ICM development and contact site formation at alphaproteobacterial envelopes. Overall, we provide support for the hypothesis that mitochondrial cristae evolved from alphaproteobacterial ICMs, and therefore have improved our understanding of the nature of the mitochondrial ancestor.

A Cristae-Like Microcompartment in Desulfobacterota

“…It has been proposed that cristae evolved from Alphaproteobacterial ICMs ( 9 , 20 , 21 , 23 ). However, D. carbinolicus and the presumed ANME-partner Desulfobacterota SRB studied here display pera that more closely resemble opisthokont cristae than Alphaproteobacterial ICMs.…”

“…And, is there even an equivalent to the MICOS in the cells that we studied? Mic60 (mitofilin domain-containing) is phylogenetically restricted to the Alphaproteobacteria and mitochondria ( 23 , 59 , 60 ) and is without homologs of the key protein components involved in cristae formation in Desulfobacterota (i.e., Mic60, 10, 12, 19, 26, and other homologs) ( Table S1 ). Desulfobacterota may have convergently evolved a structure similar to that found in opisthokont mitochondria.…”

“…It has been hypothesized that ICM junctions compartmentalize bioenergetics by restricting the diffusion of metabolites, by localizing proton gradients, and by localizing particular membrane proteins to the ICM ( 9 ). In Alphaproteobacteria , ICM-afforded compartmentalization is thought to be facilitated by the equivalent of the mitochondrial contact site and cristae organizing system (MICOS) ( 9 , 21 – 23 ). MICOS subunits in mitochondria, all with the base name Mic, are anchored at the mouth of the crista junction and, together with the soluble Opa1 protein, they form a cap at the crista junction mouth that can be opened by certain stimuli, such as apoptotic signals ( 6 ).…”

“…A stabilizing subunit, Mic60, is a core anchor in the membrane of the crista junction mouth that recruits other subunits that form the junctional scaffold ( 6 ). There is direct experimental evidence for the involvement of alphaproteobacterial-derived Mic60 in photosynthetic ICM development ( 23 ). Because ICMs have been observed without ICM junctions in the form of un-attached “stacks” of membranes, ( 11 ), ICM junctions may not be always essential to the maintenance of ICMs, unlike the crucial role the crista junction plays in the formation and function of crista ( 22 ).…”

“…The observation of ICMs in Alphaproteobacteria and the identification of proteins involved in their formation, some with homology to those in opisthokont mitochondria, led to the hypothesis that Alphaproteobacteria ICMs are the progenitor of the mitochondrial cristae ( 9 , 20 , 21 , 23 ). Outside the Alphaproteobacteria , though, as discussed above, ICMs are common in several bacterial lineages and are affiliated with specific metabolic functions rather than taxonomic bacterial groups (i.e., ammonia and nitrite oxidation, methane oxidation, photosynthesis).…”