Mechanisms of ER Protein Import

Protein import into the endoplasmic reticulum (ER) is the first step in the biogenesis of approximately 10,000 different soluble and membrane proteins of human cells, which amounts to about 30% of the proteome. Most of these proteins fulfill their functions either in the membrane or lumen of the ER plus the nuclear envelope, in one of the organelles of the pathways for endo- and exocytosis (ERGIC, Golgi apparatus, endosome, lysosome, trafficking vesicles), or at the cell surface as the plasma membrane or secreted proteins. In addition, an increasing number of membrane proteins destined for lipid droplets, peroxisomes or mitochondria are observed to be first targeted to and inserted into the ER membrane prior to their integration into budding lipid droplets or peroxisomes or prior to their delivery to mitochondria via the ER-SURF pathway. ER protein import involves two stages, ER targeting, which guarantees organelle specificity, and insertion of nascent membrane proteins into or translocation of soluble precursor polypeptides across the ER membrane. In most cases, both processes depend on amino-terminal signal peptides or transmembrane helices, which serve as targeting equivalents. However, the targeting reaction can also involve the ER targeting of specific mRNAs or ribosome-nascent chain complexes. In addition, both processes are facilitated by various sophisticated machineries, which reside in the cytosol and the ER membrane, respectively. Except for resident ER, nuclear and mitochondrial membrane proteins, the mature proteins are delivered to their functional locations by vesicular transport. In this Special Issue, renowned international experts in this area of cell biology report on their structural and mechanistic insights into various aspects of targeting, insertion, and translocation machineries, such as the signal recognition particle (SRP), its corresponding receptor (SR) and the Sec61 complex. M. Pool [1] provides a timely overview about the different pathways for targeting of soluble and membrane proteins to the ER and the triage that is taking place in the cytosol and guarantees delivery of newly synthesized polypep tides to the correct organelle, folding in the cytosol or degradation by the proteasome. H-.H. Hsieh together with S.-ou Shan [2] describe in their chapter the recent progress in deciphering the molecular mechanisms of the paradigm SRP-dependent targeting pathway and the key role of the cytosolic chaperone NAC in preventing protein mistargeting to the ER. As further reading on this subject, we suggest recent original articles by Jomaa et al. [3] as well as Tirincsi et al. [4]. J. Herrmann and colleagues [5] and B. Schrul and colleagues [6] round up the section on protein targeting to the ER and provide a state of the art view of the ER-SURF pathway and recent insights into the client spectrum of the PEX3/PEX19 pathway to the ER, respectively. Here, we suggest as further reading an article on the targeting of mRNAs and ribosome-nascent-chain complexes to the ER [7]. The next section of the Special Issue deals with the machineries for membrane insertion and translocation of proteins in the ER membrane with special emphasis on the central role of the Sec61 complex. Here, A. Tirincsi et al. [8] provide up to date insights into the connections between the different targeting and translocation/insertion machineries, while P. Bhadra and V. Helms [9] as well as M. Liaci and F. Förster [10] focus on molecular dynam ics and structural aspects of the Sec61 complex, respectively. This part of the Special Issue is finished off by S.-j. Jung together with H. Kim [11] and P. Whitley et al. [12], who discuss the current views on the Sec62/Sec63 complex in protein translocation and the biophysics of folding and insertion of transmembrane helices at the ER membrane, respectively. As further reading on the structures and molecular mechanisms of additional ER membrane resident membrane protein invertases, we suggest the excellent recent reviews by Borgese et al. [13], O´Keefe et al. [14], and Hegde and Keenan [15]. Last but not least, small molecule inhibitors and toxins that interfere with ER protein import are discussed by K. Vermeire and colleagues [16], thereby providing a link to human medicine, specifically to the so-called Sec61-channelopathies. This medical aspect is discussed by M. Pool [1] and was recently reviewed by Sicking et al. [17]. Two original articles complete the Special Issue, the one by E. Pauwels et al. [18] on the mode of action of the ER protein import inhibitor cyclotri azadisulfonamide (CADA), and the other one by M. Sicking et al. [19] on the adaption of a novel bimolecular luminescence approach to the analysis of the dynamics of ER membrane components, which are in volved in targeting, translocation and membrane insertion of polypeptides at the ER of living human cells

AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma.

AHNAK/Desmoyokin is a giant protein which has been recently linked to reorganization of the actin cytoskeleton, cellular migration and invasion. Here, we investigated the role of AHNAK in the pathophysiology of larynx carcinoma-one of the major subtypes of head and neck cancer. To this end, we analysed AHNAK expression in tumor tissues from 83 larynx carcinoma patients in relation to overall survival. We found that tumoral AHNAK overexpression significantly associated with poor survival of these patients both in univariate and multivariate analysis. In further studies, we combined the prognostic value of AHNAK with selected markers of inflammation, such as macrophage migration inhibitory factor (MIF) and tumor-infiltrating neutrophils (CD66b-positive cells). Both MIF and neutrophils have been linked to enhanced tumoral migration and poor clinical outcome in patients with orohypopharynx carcinoma-another major subtype of head and neck cancer. Interestingly, we found that synchronous high levels of AHNAK and MIF or AHNAK and neutrophils, respectively, were stronger predictors of poor survival than AHNAK alone. Synchronous high levels of all three markers were the strongest predictors of poor survival in our patient cohort. Taken together, our findings propose novel strategies for an accurate prognosis in larynx carcinoma and suggest potential mechanisms of inflammation-mediated tumor progression

A least squares approach to reduce stable discrete linear systems preserving their stability

AbstractA new stability preserving model reduction algorithm for discrete linear SISO-systems based on a least squares approach is proposed. Similar to the Padé approximation, an equation system for the Markov parameters involving a high dimensional Hankel matrix is considered. It is proved that approximate solutions, computed via the Moore–Penrose pseudo-inverse, give rise to a stability preserving reduction scheme. Furthermore, the proposed algorithm is compared to the balanced truncation method, showing comparable performance of the reduced systems

Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells

Does Gibrat’s Law Hold for Retailing? Evidence from Sweden

Gibrat’s Law predicts that firm growth is a purely random effect and therefore should be independent of firm size. The purpose of this paper is to test Gibrat’s law within the retail industry, using a novel data-set comprising all Swedish limited liability companies active at some point between 1998 and 2004. Very few studies have previously investigated whether Gibrat’s Law seems to hold for retailing, and they are based on highly aggregated data. Our results indicate that Gibrat´s Law can be rejected for a large majority of five-digit retail industries in Sweden, since small retail firms tend to grow faster than large ones.firm dynamics; firm size; firm growth; retail

Towards efficient structure prediction and pre-compensation in multi-photon lithography

Microscale 3D printing technologies have been of increasing interest in industry and research for several years. Unfortunately, the fabricated structures always deviate from the respective expectations, often caused by the physico-chemical properties during and after the printing process. Here, we show first steps towards a simple, fast and easy to implement algorithm to predict the final structure topography for multi-photon lithography - also known as Direct Laser Writing (DLW). The three main steps of DLW, (i) exposure of a photo resin, (ii) cross-linking of the resin, and (iii) subsequent shrinkage are approximated by mathematical operations, showing promising results in coincidence with experimental observations. E.g., the root-mean-square error (rmse) between the unmodified 3D print of a radial-symmetrically chirped topography and our predicted topography is only 0.46 $\mu$m, whereas the rmse between this 3D print and its target is 1.49 $\mu$m. Thus, our robust predictions can be used prior to the printing process to minimize undesired deviations between the target structure and the final 3D printed structure. Using a Downhill-Simplex algorithm for identifying the optimal prediction parameters, we were able to reduce the rmse from 4.04 $\mu$m to 0.33 $\mu$m by only two correction loops in our best-case scenario (rmse = 0.72 $\mu$m after one loop). Consequently, this approach can eliminate the need for many structural optimization loops to produce highly conformal and high quality micro structures in the future

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components

ER import of small human presecretory proteins: components and mechanisms

Protein transport into the mammalian endoplasmic reticulum (ER) used to be seen as strictly cotranslational, that is temporarily and mechanistically coupled to protein synthesis. In the course of the last decades, however, several classes of precursors of soluble and membrane proteins were found to be post-translationally imported into the ER, without any involvement of the ribosome. The first such class to be identified were the small presecretory proteins; tail-anchored membrane proteins followed next. In both classes, the inherent address tag is released from the translating ribosome before the initiation of ER import, as part of the fully synthesized precursor. In small presecretory proteins, the information for ER targeting and -translocation via the polypeptide-conducting Sec61-channel is encoded by a classical N-terminal signal peptide, which is released from the ribsosome before targeting due to the small size of the full-length precursor. Here, we discuss the current state of research on targeting and translocation of small presecretory proteins into the mammalian ER. In closing, we present a unifying hypothesis for ER protein translocation in terms of an energy diagram for Sec61-channel gating