Coordinated Translocation of Presequence-Containing Precursor Proteins Across Two Mitochondrial Membranes: Knowns and Unknowns of How TOM and TIM23 Complexes Cooperate With Each Other

The vast majority of mitochondrial proteins are encoded in the nuclear genome and synthesized on cytosolic ribosomes as precursor proteins with specific mitochondrial targeting signals. Mitochondrial targeting signals are very diverse, however, about 70% of mitochondrial proteins carry cleavable, N-terminal extensions called presequences. These amphipathic helices with one positively charged and one hydrophobic surface target proteins to the mitochondrial matrix with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. Translocation of proteins across the two mitochondrial membranes does not take place independently of each other. Rather, in the intermembrane space, where the two complexes meet, components of the TOM and TIM23 complexes form an intricate network of protein–protein interactions that mediates initially transfer of presequences and then of the entire precursor proteins from the outer to the inner mitochondrial membrane. In this Mini Review, we summarize our current understanding of how the TOM and TIM23 complexes cooperate with each other and highlight some of the future challenges and unresolved questions in the field.

Multiple Precursor Proteins of Thanatin Isoforms, an Antimicrobial Peptide Associated With the Gut Symbiont of Riptortus pedestris

Thanatin is an antimicrobial peptide (AMP) generated by insects for defense against bacterial infections. In the present study, we performed cDNA cloning of thanatin and found the presence of multiple precursor proteins from the bean bug, Riptortus pedestris. The cDNA sequences encoded 38 precursor proteins, generating 13 thanatin isoforms. In the phylogenetic analysis, thanatin isoforms were categorized into two groups based on the presence of the membrane attack complex/perforin (MACPF) domain. In insect-bacterial symbiosis, specific substances are produced by the immune system of the host insect and are known to modulate the symbiont’s population. Therefore, to determine the biological function of thanatin isoforms in symbiosis, the expression levels of three AMP genes were compared between aposymbiotic insects and symbiotic R. pedestris. The expression levels of the thanatin genes were significantly increased in the M4 crypt, a symbiotic organ, of symbiotic insects upon systemic bacterial injection. Further, synthetic thanatin isoforms exhibited antibacterial activity against gut-colonized Burkholderia symbionts rather than in vitro-cultured Burkholderia cells. Interestingly, the suppression of thanatin genes significantly increased the population of Burkholderia gut symbionts in the M4 crypt under systemic Escherichia coli K12 injection. Overgrown Burkholderia gut symbionts were observed in the hemolymph of host insects and exhibited insecticidal activity. Taken together, these results suggest that thanatin of R. pedestris is a host-derived symbiotic factor and an AMP that controls the population of gut-colonized Burkholderia symbionts.

Protein Quality Control at the Mitochondrial Surface

Mitochondria contain two membranes, the outer and inner membrane. The outer membrane fulfills crucial functions for the communication of mitochondria with the cellular environment like exchange of lipids via organelle contact sites, the transport of metabolites and the formation of a signaling platform in apoptosis and innate immunity. The translocase of the outer membrane (TOM complex) forms the entry gate for the vast majority of precursor proteins that are produced on cytosolic ribosomes. Surveillance of the functionality of outer membrane proteins is critical for mitochondrial functions and biogenesis. Quality control mechanisms remove defective and mistargeted proteins from the outer membrane as well as precursor proteins that clog the TOM complex. Selective degradation of single proteins is also an important mode to regulate mitochondrial dynamics and initiation of mitophagy pathways. Whereas inner mitochondrial compartments are equipped with specific proteases, the ubiquitin-proteasome system is a central player in protein surveillance on the mitochondrial surface. In this review, we summarize our current knowledge about the molecular mechanisms that govern quality control of proteins at the outer mitochondrial membrane.

Mapping protein interactions in the active TOM-TIM23 supercomplex

Nuclear-encoded mitochondrial proteins destined for the matrix have to be transported across two membranes. The TOM and TIM23 complexes facilitate the transport of precursor proteins with N-terminal targeting signals into the matrix. During transport, precursors are recognized by the TIM23 complex in the inner membrane for handover from the TOM complex. However, we have little knowledge on the organization of the TOM-TIM23 transition zone and on how precursor transfer between the translocases occurs. Here, we have designed a precursor protein that is stalled during matrix transport in a TOM-TIM23-spanning manner and enables purification of the translocation intermediate. Combining chemical cross-linking with mass spectrometric analyses and structural modeling allows us to map the molecular environment of the intermembrane space interface of TOM and TIM23 as well as the import motor interactions with amino acid resolution. Our analyses provide a framework for understanding presequence handover and translocation during matrix protein transport.

Influence of Switchgrass TDIF-like Genes on Arabidopsis Vascular Development

As a member of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE) family, the dodecapeptide tracheary element differentiation inhibitory factor (TDIF) has a major impact on vascular development in plants. However, the influence of polymorphisms in the TDIF peptide motif on activity remains poorly understood. The model plant, Arabidopsis provides a fast and effective tool for assaying the activity of TDIF homologs. Five TDIF homologs from a group of 93 CLE genes in switchgrass (Panicum virgatum), a perennial biomass crop, named PvTDIF-like (PvTDIFL) genes were studied. The expression levels of PvTDIFL1, PvTDIFL3MR3, and PvTDIFL3MR2 were relatively high and all of them were expressed at the highest levels in the rachis of switchgrass. The precursor proteins for PvTDIFL1, PvTDIFL3MR3, and PvTDIFL3MR2 contained one, three, and two TDIFL motifs, respectively. Treatments with exogenous PvTDIFL peptides increased the number of stele cells in the hypocotyls of Arabidopsis seedlings, with the exception of PvTDIFL_4p. Heterologous expression of PvTDIFL1 in Arabidopsis strongly inhibited plant growth, increased cell division in the vascular tissue of the hypocotyl, and disrupted the cellular organization of the hypocotyl. Although heterologous expression of PvTDIFL3MR3 and PvTDIFL3MR2 also affected plant growth and vascular development, PvTDIFL activity was not enhanced by the multiple TDIFL motifs encoded by PvTDIFL3MR3 and PvTDIFL3MR2. These data indicate that in general, PvTDIFLs are functionally similar to Arabidopsis TDIF but that the processing and activities of the PvTDIFL peptides are more complex.

Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

ER-SURF: Riding the Endoplasmic Reticulum Surface to Mitochondria

Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1) and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation (ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the current knowledge about the cooperation of the ER and mitochondria in the targeting and quality control of mitochondrial precursor proteins.

Quality control of protein import into mitochondria

Mitochondria import about 1000 proteins that are produced as precursors on cytosolic ribosomes. Defects in mitochondrial protein import result in the accumulation of non-imported precursor proteins and proteotoxic stress. The cell is equipped with different quality control mechanisms to monitor protein transport into mitochondria. First, molecular chaperones guide unfolded proteins to mitochondria and deliver non-imported proteins to proteasomal degradation. Second, quality control factors remove translocation stalled precursor proteins from protein translocases. Third, protein translocases monitor protein sorting to mitochondrial subcompartments. Fourth, AAA proteases of the mitochondrial subcompartments remove mislocalized or unassembled proteins. Finally, impaired efficiency of protein transport is an important sensor for mitochondrial dysfunction and causes the induction of cellular stress responses, which could eventually result in the removal of the defective mitochondria by mitophagy. In this review, we summarize our current understanding of quality control mechanisms that govern mitochondrial protein transport.

Peptidomic Analysis of Neonate Umbilical Cord Blood for the Identification of Endogenous Peptides Involved in Hypoxic–Ischemic Encephalopathy

Neonatal hypoxic–ischemic encephalopathy (HIE) is a common neurological disorder triggered by perinatal cerebral ischemia and hypoxia. Accumulating evidence has shown that peptides have neuroprotective effects in nerve injury. However, the function of endogenous peptides in the pathogenesis of HIE has not been studied. In the present study, a comparative peptidomic profile was performed in the serum of the human umbilical cord blood with HIE (three patients) and the control group (three health control) by liquid chromatography–mass spectrometry (LC-MS). Our study demonstrated that a total of 49 peptides derived from 25 precursor proteins were differentially expressed in the serum of HIE compared with normal controls, including 33 upregulated peptides and 16 downregulated peptides. Each of the differentially expressed peptides has specific characteristics, including pI, Mw, and cleavage pattern. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that the precursor proteins of differentially expressed peptides participate in the different biological process. Moreover, among the 49 differentially expressed peptides, 21 peptides were identified from the fibrinogen chain family, which plays a role in neurological diseases, suggesting that these peptides may play an important role in maintaining brain health. In conclusion, our results showed a comparative peptidomic profile from human umbilical cord blood of HIE patients and normal controls. These dysregulated peptides may have potentially important functions in umbilical cord blood with HIE and may be involved in the pathogenesis of the HIE.