Ubiquitin profiling of lysophagy identifies actin stabilizer CNN2 as a target of VCP/p97 and uncovers a link to HSPB1.

Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.

Unveiling ubiquitinome rearrangements induced by Salmonella infection.

Ubiquitination plays a critical role in the activation of host immune responses to infection and serves as a signal for pathogen delivery to phagophores along the xenophagy pathway. We recently performed systematic ubiquitination site profiling of epithelial cells infected with Salmonella Typhimurium. Our findings specifically highlight components of the NFKB, membrane trafficking pathways and RHO GTPase systems as ubiquitination hubs during infection. In addition, a broad spectrum of bacterial effectors and several outer membrane proteins are ubiquitinated in infected cells. This comprehensive resource of ubiquitinome dynamics during Salmonella infection enables further understanding of the complex host-pathogen interplay and may reveal novel targets for the inhibition of Salmonella invasion and inflammation.

Global Analysis of Host and Bacterial Ubiquitinome in Response to Salmonella Typhimurium Infection.

Ubiquitination serves as a critical signal in the host immune response to infection. Many pathogens have evolved strategies to exploit the ubiquitin (Ub) system to promote their own survival through a complex interplay between host defense machinery and bacterial virulence factors. Here we report dynamic changes in the global ubiquitinome of host epithelial cells and invading pathogen in response to Salmonella Typhimurium infection. The most significant alterations in the host ubiquitinome concern components of the actin cytoskeleton, NF-κB and autophagy pathways, and the Ub and RHO GTPase systems. Specifically, infection-induced ubiquitination promotes CDC42 activity and linear ubiquitin chain formation, both being required for NF-κB activation. Conversely, the bacterial ubiquitinome exhibited extensive ubiquitination of various effectors and several outer membrane proteins. Moreover, we reveal that bacterial Ub-modifying enzymes modulate a unique subset of host targets, affecting different stages of Salmonella infection.

Eukaryotic Hsp70 chaperones in the intermembrane space of chloroplasts.

MAIN CONCLUSION: Multiple eukaryotic Hsp70 typically localized in the cytoplasm are also distributed to the intermembrane space of chloroplasts and might thereby represent the missing link in energizing protein translocation. Protein translocation into organelles is a central cellular process that is tightly regulated. It depends on signals within the preprotein and on molecular machines catalyzing the process. Molecular chaperones participate in transport and translocation of preproteins into organelles to control folding and to provide energy for the individual steps. While most of the processes are explored and the components are identified, the transfer of preproteins into and across the intermembrane space of chloroplasts is not yet understood. The existence of an energy source in this compartment is discussed, because the required transit peptide length for successful translocation into chloroplasts is shorter than that found for mitochondria where energy is provided exclusively by matrix chaperones. Furthermore, a cytosolic-type Hsp70 homologue was proposed as component of the chloroplast translocon in the intermembrane space energizing the initial translocation. The molecular identity of such intermembrane space localized Hsp70 remained unknown, which led to a controversy concerning its existence. We identified multiple cytosolic Hsp70s by mass spectrometry on isolated, thermolysin-treated Medicago sativa chloroplasts. The localization of these Hsp70s of M. sativa or Arabidopsis thaliana in the intermembrane space was confirmed by a self-assembly GFP-based in vivo system. The localization of cytosolic Hsp70s in the stroma of chloroplasts or different mitochondrial compartments could not be observed. Similarly, we could not identify any cytosolic Hsp90 in the intermembrane space of chloroplast. With respect to our results we discuss the possible targeting and function of the Hsp70 found in the intermembrane space.

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Chloroplast import signals: the length requirement for translocation in vitro and in vivo.

Protein translocation of cytosolically synthesized proteins requires signals for both targeting of precursor proteins to the surface of the respective compartment and their transfer across its membrane. In contrast to signals for peroxisomal and endoplasmic reticulum translocation, the signals for mitochondrial and chloroplast transport are less well defined with respect to length and amino acid requirements. To study the properties of signals required for translocation into chloroplasts in vitro and in vivo, we used fusion proteins composed of transit peptides and the Ig-like module of the muscle protein titin as passenger. We observed that about 60 amino acids-longer than the transit peptide length of many experimentally confirmed chloroplast proteins-are required for efficient translocation. However, within native chloroplast precursor proteins with transit peptides shorter than 60 amino acids, extension appears to be present as they are efficiently imported into organelles. In addition, the interaction of an unfolded polypeptide stretch of 60 or more amino acids with receptors at the chloroplast surface results in the unidirectionality of protein translocation into chloroplasts even in the presence of a competing C-terminal peroxisomal targeting signal. These findings prove the existing ideas that initial targeting is defined by the N-terminal signal and that the C-terminal signal is sensed only subsequently.

On the impact of precursor unfolding during protein import into chloroplasts.

Protein translocation across membranes is a fundamental cellular process. The majority of the proteins of organelles such as mitochondria and chloroplasts is synthesized in the cytosol and subsequently imported in a post-translational manner. The precursor proteins have to be unfolded at least for translocation, but it has also been assumed that they are unfolded during transport to the organelle in the cytosol. Unfolding is governed by chaperones and the translocon itself. At the same time, chaperones provide the energy for the import process. The energetic properties of the chloroplast translocon were studied by import of the Ig-like module of the muscle protein titin fused to the transit peptide of the chloroplast targeted oxygen evolving complex subunit of 33 kDa (OE33). Our results suggest that p(OE33)titin is folded prior to import and that translocation is initiated by unfolding after having bound to the translocon at the chloroplast surface. Using a set of stabilizing and destabilizing mutants of titin previously analyzed by atomic force microscopy and as passenger for mitochondrial translocation, we studied the unfolding force provided by the chloroplast translocon. Based on these results, a model for translocation is discussed.

Tethering of ferredoxin:NADP+ oxidoreductase to thylakoid membranes is mediated by novel chloroplast protein TROL.

Working in tandem, two photosystems in the chloroplast thylakoid membranes produce a linear electron flow from H(2)O to NADP(+). Final electron transfer from ferredoxin to NADP(+) is accomplished by a flavoenzyme ferredoxin:NADP(+) oxidoreductase (FNR). Here we describe TROL (thylakoid rhodanese-like protein), a nuclear-encoded component of thylakoid membranes that is required for tethering of FNR and sustaining efficient linear electron flow (LEF) in vascular plants. TROL consists of two distinct modules; a centrally positioned rhodanese-like domain and a C-terminal hydrophobic FNR binding region. Analysis of Arabidopsis mutant lines indicates that, in the absence of TROL, relative electron transport rates at high-light intensities are severely lowered accompanied with significant increase in non-photochemical quenching (NPQ). Thus, TROL might represent a missing thylakoid membrane docking site for a complex between FNR, ferredoxin and NADP(+). Such association might be necessary for maintaining photosynthetic redox poise and enhancement of the NPQ.

Evolutionarily evolved discriminators in the 3-TPR domain of the Toc64 family involved in protein translocation at the outer membrane of chloroplasts and mitochondria.

Transport of polypeptides across membranes is a general and essential cellular process utilised by molecular machines. At least one component of these complexes contains a domain composed of three tetratricopeptide repeat (3-TPR) motifs. We have focussed on the receptor Toc64 to elucidate the evolved functional specifications of its 3-TPR domain. Toc64 is a component of the Toc core complex and functionally replaces Tom70 at the outer membrane of mitochondria in plants. Its 3-TPR domain recognises the conserved C-terminus of precursor-bound chaperones. We built homology models of the 3-TPR domain of chloroplastic Toc64 from different species and of the mitochondrial isoform from Arabidopsis. Guided by modelling, we identified residues essential for functional discrimination of the differently located isoforms to be located almost exclusively on the convex surface of the 3-TPR domain. The only exception is at568Ser/ps557Met, which is positioned in the ligand-binding groove. The functional implications of the homology models are discussed.

pH sensitivity of the GTPase Toc33 as a regulatory circuit for protein translocation into chloroplasts.

The properties of membrane-embedded GTPases are investigated to understand translocation of preprotein across the outer envelope of chloroplasts. The homo- and heterodimerization events of the GTPases had been established previously. We show that the hydrolytic activity of the GTPase Toc33 is pH insensitive in the homodimeric conformation but has a bell-shaped pH optimum in the monomeric conformation. Further, Toc33 GTPase homodimerization and protein translocation into chloroplasts are pH sensitive as well. pH sensitivity might serve to regulate translocation; alternatively, the documented pH sensitivity might reflect a mechanistic requirement for GTPase silencing during translocation as the GTPase switches between homo- and heterodimeric conformations.

Toc64--a preprotein-receptor at the outer membrane with bipartide function.

Protein translocation across membranes is assisted by translocation machineries present in the membrane targeted by the precursor proteins. Translocon subunits can be functionally divided into receptor proteins warranting the specificity of this machine and a translocation channel. At the outer envelope of chloroplasts two sets of receptor proteins regulate protein translocation facing the cytosol or acting in the intermembrane space. One, Toc64 is a receptor of the translocon at the outer envelope of chloroplasts (Toc complex) with dual function. Toc64 recognizes Hsp90 delivered precursor proteins via a cytosolic exposed domain containing three tetratrico-peptide repeat motifs and as demonstrated in here, Toc64 functions also as a major component of a complex facing the intermembrane space. The latter complex is composed of an Hsp70 localized in the intermembrane space, its interaction partner Toc12, a J-domain containing protein and the intermembrane space protein Tic22. We analyzed the intermembrane space domain of Toc64. This domain is involved in preprotein recognition and association with the Toc-complex independent of the cytosolic domain of the Toc64 receptor. Therefore, Toc64 is involved in preprotein translocation across the outer envelope at both sites of the membrane.