Goran Stjepanovic

Escherichia coli Signal Recognition Particle Receptor FtsY Contains an Essential and Autonomous Membrane-binding Amphipathic Helix

Escherichia coli membrane protein biogenesis is mediated by a signal recognition particle and its membrane-associated receptor (FtsY). Although crucial for its function, it is still not clear how FtsY interacts with the membrane. Analysis of the structure/function differences between severely truncated active (NG+1) and inactive (NG) mutants of FtsY enabled us to identify an essential membrane-interacting determinant. Comparison of the three-dimensional structures of the mutants, combined with site-directed mutagenesis, modeling, and liposome-binding assays, revealed that FtsY contains a conserved autonomous lipid-binding amphipathic α-helix at the N-terminal end of the N domain. Deletion experiments showed that this helix is essential for FtsY function in vivo, thus offering, for the first time, clear evidence for the functionally important, physiologically relevant interaction of FtsY with lipids.

Structural insights into tail-anchored protein binding and membrane insertion by Get3

Tail-anchored (TA) membrane proteins are involved in a variety of important cellular functions, including membrane fusion, protein translocation, and apoptosis. The ATPase Get3 (Asna1, TRC40) was identified recently as the endoplasmic reticulum targeting factor of TA proteins. Get3 consists of an ATPase and α-helical subdomain enriched in methionine and glycine residues. We present structural and biochemical analyses of Get3 alone as well as in complex with a TA protein, ribosome-associated membrane protein 4 (Ramp4). The ATPase domains form an extensive dimer interface that encloses 2 nucleotides in a head-to-head orientation and a zinc ion. Amide proton exchange mass spectrometry shows that the α-helical subdomain of Get3 displays considerable flexibility in solution and maps the TA protein-binding site to the α-helical subdomain. The non-hydrolyzable ATP analogue AMPPNP-Mg2+- and ADP-Mg2+-bound crystal structures representing the pre- and posthydrolysis states are both in a closed form. In the absence of a TA protein cargo, ATP hydrolysis does not seem to be possible. Comparison with the ADP·AlF4−-bound structure representing the transition state (Mateja A, et al. (2009) Nature 461:361–366) indicates how the presence of a TA protein is communicated to the ATP-binding site. In vitro membrane insertion studies show that recombinant Get3 inserts Ramp4 in a nucleotide- and receptor-dependent manner. Although ATP hydrolysis is not required for Ramp4 insertion per se, it seems to be required for efficient insertion. We postulate that ATP hydrolysis is needed to release Get3 from its receptor. Taken together, our results provide mechanistic insights into posttranslational targeting of TA membrane proteins by Get3.

Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) that functions in early autophagy consists of the lipid kinase VPS34, the scaffolding protein VPS15, the tumor suppressor BECN1, and the autophagy-specific subunit ATG14. The structure of the ATG14-containing PI3KC3-C1 was determined by single-particle EM, revealing a V-shaped architecture. All of the ordered domains of VPS34, VPS15, and BECN1 were mapped by MBP tagging. The dynamics of the complex were defined using hydrogen–deuterium exchange, revealing a novel 20-residue ordered region C-terminal to the VPS34 C2 domain. VPS15 organizes the complex and serves as a bridge between VPS34 and the ATG14:BECN1 subcomplex. Dynamic transitions occur in which the lipid kinase domain is ejected from the complex and VPS15 pivots at the base of the V. The N-terminus of BECN1, the target for signaling inputs, resides near the pivot point. These observations provide a framework for understanding the allosteric regulation of lipid kinase activity. DOI: http://dx.doi.org/10.7554/eLife.05115.001

Synchronizing nuclear import of ribosomal proteins with ribosome assembly.

Symportin Synchrony Ribosomes, the macromolecular machines responsible for protein synthesis, function in the cytoplasm but are assembled in the nucleus. Ribosomal proteins must be imported into the nucleus, but how this is coordinated with assembly is unclear. Kressler et al. (p. 666) report that two 5S rRNA binding proteins are coimported into the nucleus. They identify a transport adaptor, which they term symportin (Syo1), that binds simultaneously to Rpl5 and Rpl11. Syo1 also interacts with the import receptor Kap104, which facilitates import of the Syo1-Rpl5-Rpl11 complex. Synchronous nuclear transport may be more generally used to coordinate assembly processes. The transport adaptor symportin mediates stoichiometric import of a pair of ribosomal proteins. Ribosomal proteins are synthesized in the cytoplasm, before nuclear import and assembly with ribosomal RNA (rRNA). Little is known about coordination of nucleocytoplasmic transport with ribosome assembly. Here, we identify a transport adaptor, symportin 1 (Syo1), that facilitates synchronized coimport of the two 5S-rRNA binding proteins Rpl5 and Rpl11. In vitro studies revealed that Syo1 concomitantly binds Rpl5-Rpl11 and furthermore recruits the import receptor Kap104. The Syo1-Rpl5-Rpl11 import complex is released from Kap104 by RanGTP and can be directly transferred onto the 5S rRNA. Syo1 can shuttle back to the cytoplasm by interaction with phenylalanine-glycine nucleoporins. X-ray crystallography uncovered how the α-solenoid symportin accommodates the Rpl5 amino terminus, normally bound to 5S rRNA, in an extended groove. Symportin-mediated coimport of Rpl5-Rpl11 could ensure coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

The Crystal Structure of the Periplasmic Domain of the Escherichia coli Membrane Protein Insertase YidC Contains a Substrate Binding Cleft

In bacteria the biogenesis of inner membrane proteins requires targeting and insertion factors such as the signal recognition particle and the Sec translocon. YidC is an essential membrane protein involved in the insertion of inner membrane proteins together with the Sec translocon, but also as a separate entity. YidC of Escherichia coli is a member of the conserved YidC (in bacteria)/Oxa1 (in mitochondria)/Alb3 (in chloroplasts) protein family and contains six transmembrane segments and a large periplasmic domain (P1). We determined the crystal structure of the periplasmic domain of YidC from E. coli (P1D) at 1.8 Å resolution. The structure of P1D shows the conserved β-supersandwich fold of carbohydrate-binding proteins and an α-helical linker region at the C terminus that packs against the β-supersandwich by a highly conserved interface. P1D exhibits an elongated cleft of similar architecture as found in the structural homologs. However, the electrostatic properties and molecular details of the cleft make it unlikely to interact with carbohydrate substrates. The cleft in P1D is occupied by a polyethylene glycol molecule suggesting an elongated peptide or acyl chain as a natural ligand. The region of P1D previously reported to interact with SecF maps to a surface area in the vicinity of the cleft. The conserved C-terminal region of the P1 domain was reported to be essential for the membrane insertase function of YidC. The analysis of this region suggests a role in membrane interaction and/or in the regulation of YidC interaction with binding partners.

Vps4 disassembles an ESCRT-III filament by global unfolding and processive translocation

The AAA+ ATPase Vps4 disassembles ESCRT-III and is essential for HIV-1 budding and other pathways. Vps4 is a paradigmatic member of a class of hexameric AAA+ ATPases that disassemble protein complexes without degradation. To distinguish between local displacement versus global unfolding mechanisms for complex disassembly, we carried out hydrogen/deuterium exchange during Saccharomyces cerevisiae Vps4 disassembly of a chimeric Vps24-2 ESCRT-III filament. EX1 exchange behavior shows that Vps4 completely unfolds ESCRT-III substrates on a time scale consistent with the disassembly reaction. The established unfoldase ClpX showed the same pattern, thus demonstrating a common unfolding mechanism. Vps4 hexamers containing a single cysteine residue in the pore loops were cross-linked to ESCRT-III subunits containing unique cysteines within the folded core domain. These data support a mechanism in which Vps4 disassembles its substrates by completely unfolding them and threading them through the central pore.

Membrane Targeting of Ribosomes and Their Release Require Distinct and Separable Functions of FtsY

The mechanism underlying the interaction of the Escherichia coli signal recognition particle (SRP) receptor FtsY with the cytoplasmic membrane is not fully understood. We investigated this issue by utilizing active (NG+1) and inactive (NG) mutants of FtsY. In solution, the mutants comparably bind and hydrolyze nucleotides and associate with SRP. In contrast, a major difference was observed in the cellular distribution of NG and NG+1. Unlike NG+1, which distributes almost as the wild-type receptor, the inactive NG mutant accumulates on the membrane, together with ribosomes and SRP. The results suggest that NG function is compromised only at a later stage of the targeting pathway and that despite their identical behavior in solution, the membrane-bound NG-SRP complex is less active than NG+1-SRP. This notion is strongly supported by the observation that lipids stimulate the GTPase activity of NG+1-SRP, whereas no stimulation is observed with NG-SRP. In conclusion, we propose that the SRP receptor has two distinct and separable roles in (i) mediating membrane targeting and docking of ribosomes and (ii) promoting their productive release from the docking site.

Assembly and dynamics of the autophagy-initiating Atg1 complex

Significance Autophagy is conserved and essential for cellular survival during starvation and stress, and is initiated by the autophagy-related 1 (Atg1) complex. In yeast, the three subunits Atg17, Atg29, and Atg31 assemble first, and their structure is known. There has been a debate over how the Atg1 and Atg13 subunits assemble when autophagy is triggered. Here we use mass spectrometry to show that the C-terminal domain of Atg1 is highly dynamic on its own. Atg1 forms a stable complex when it binds with high affinity to Atg13. The combined Atg1–Atg13 subcomplex then binds with moderate affinity to the preformed Atg17–Atg31–Atg29 scaffold. This highlights the binding of Atg1–Atg13 to Atg17–Atg31–Atg29 as a pivotal step in autophagy initiation. The autophagy-related 1 (Atg1) complex of Saccharomyces cerevisiae has a central role in the initiation of autophagy following starvation and TORC1 inactivation. The complex consists of the protein kinase Atg1, the TORC1 substrate Atg13, and the trimeric Atg17–Atg31–Atg29 scaffolding subcomplex. Autophagy is triggered when Atg1 and Atg13 assemble with the trimeric scaffold. Here we show by hydrogen–deuterium exchange coupled to mass spectrometry that the mutually interacting Atg1 early autophagy targeting/tethering domain and the Atg13 central domain are highly dynamic in isolation but together form a stable complex with ∼100-nM affinity. The Atg1–Atg13 complex in turn binds as a unit to the Atg17–Atg31–Atg29 scaffold with ∼10-μM affinity via Atg13. The resulting complex consists primarily of a dimer of pentamers in solution. These results lead to a model for autophagy initiation in which Atg1 and Atg13 are tightly associated with one another and assemble transiently into the pentameric Atg1 complex during starvation.

Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex

The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners.

Lipids Trigger a Conformational Switch That Regulates Signal Recognition Particle (SRP)-mediated Protein Targeting

Co-translational protein targeting to the membrane is mediated by the signal recognition particle and its receptor (FtsY). Their homologous GTPase domains interact at the membrane and form a heterodimer in which both GTPases are activated. The prerequisite for protein targeting is the interaction of FtsY with phospholipids. However, the mechanism of FtsY regulation by phospholipids remained unclear. Here we show that the N terminus of FtsY (A domain) is natively unfolded in solution and define the complete membrane-targeting sequence. We show that the membrane-targeting sequence is highly dynamic in solution, independent of nucleotides and directly responds to the density of anionic phospholipids by a random coil-helix transition. This conformational switch is essential for tethering FtsY to membranes and activates the GTPase for its subsequent interaction with the signal recognition particle. Our results underline the dynamics of lipid-protein interactions and their importance in the regulation of protein targeting and translocation across biological membranes.