Regulation of endoplasmic reticulum-associated protein degradation (ERAD) by ubiquitin

Quality control of protein folding inside the endoplasmic reticulum (ER) includes chaperone-mediated assistance in folding and the selective targeting of terminally misfolded species to a pathway called ER-associated protein degradation, or simply ERAD. Once selected for ERAD, substrates will be transported (back) into the cytosol, a step called retrotranslocation. Although still ill defined, retrotranslocation likely involves a protein conducting channel that is in part formed by specific membrane-embedded E3 ubiquitin ligases. Early during retrotranslocation, reversible self-ubiquitination of these ligases is thought to aid in initiation of substrate transfer across the membrane. Once being at least partially exposed to the cytosol, substrates will become ubiquitinated on the cytosolic side of the ER membrane by the same E3 ubiquitin ligases. Ubiquitin on substrates was originally thought to be a permanent modification that (1) promotes late steps of retrotranslocation by recruiting the energy-providing ATPase Cdc48p/p97 via binding to its associated adaptor proteins and that (2) serves to target substrates to the proteasome. Recently it became evident, however, that the poly-ubiquitin chains (PUCs) on ERAD substrates are often subject to extensive remodeling, or processing, at several stages during ERAD. This review recapitulates the current knowledge and recent findings about PUC processing on ERAD substrates and ubiquitination of ERAD machinery components and discusses their functional consequences

Combining multiple classifications of chemical structures using consensus clustering

Consensus clustering involves combining multiple clusterings of the same set of objects to achieve a single clustering that will, hopefully, provide a better picture of the groupings that are present in a dataset. This Letter reports the use of consensus clustering methods on sets of chemical compounds represented by 2D fingerprints. Experiments with DUD, IDAlert, MDDR and MUV data suggests that consensus methods are unlikely to result in significant improvements in clustering effectiveness as compared to the use of a single clustering method. (C) 2012 Elsevier Ltd. All rights reserved

Stitching proteins into membranes, not sew simple

Most integral membrane proteins located within the endomembrane system of eukaryotic cells are first assembled co-translationally into the endoplasmic reticulum (ER) before being sorted and trafficked to other organelles. The assembly of membrane proteins is mediated by the ER translocon, which allows passage of lumenal domains through and lateral integration of transmembrane (TM) domains into the ER membrane. It may be convenient to imagine multi-TM domain containing membrane proteins being assembled by inserting their first TM domain in the correct orientation, with subsequent TM domains inserting with alternating orientations. However a simple threading model of assembly, with sequential insertion of one TM domain into the membrane after another, does not universally stand up to scrutiny. In this article we review some of the literature illustrating the complexities of membrane protein assembly. We also present our own thoughts on aspects that we feel are poorly understood. In short we hope to convince the readers that threading of membrane proteins into membranes is 'not sew simple' and a topic that requires further investigation

Limited ER quality control for GPI-anchored proteins

Endoplasmic reticulum (ER) quality control mechanisms target terminally misfolded proteins for ER-associated degradation (ERAD). Misfolded glycophosphatidylinositol-anchored proteins (GPI-APs) are, however, generally poor ERAD substrates and are targeted mainly to the vacuole/lysosome for degradation, leading to predictions that a GPI anchor sterically obstructs ERAD. Here we analyzed the degradation of the misfolded GPI-AP Gas1* in yeast. We could efficiently route Gas1* to Hrd1-dependent ERAD and provide evidence that it contains a GPI anchor, ruling out that a GPI anchor obstructs ERAD. Instead, we show that the normally decreased susceptibility of Gas1* to ERAD is caused by canonical remodeling of its GPI anchor, which occurs in all GPI-APs and provides a protein-independent ER export signal. Thus, GPI anchor remodeling is independent of protein folding and leads to efficient ER export of even misfolded species. Our data imply that ER quality control is limited for the entire class of GPI-APs, many of them being clinically relevantEspaña, Ministerio de Ciencia e Innovación BFU2014-59309-PEspaña, Ministerio de Ciencia e Innovación BFU2009-07290España, Junta de Andalucía P09-CVI-450

Protein O-mannosyltransferases participate in ER protein quality control

In eukaryotic cells, proteins enter the secretory pathway at the endoplasmic reticulum (ER) as linear polypeptides and fold after translocation across or insertion into the membrane. If correct folding fails, many proteins are O-mannosylated inside the ER by an O-mannosyltransferase, the Pmt1p-Pmt2p complex. The consequences of this modification are controversial and the cellular role of the Pmt1p-Pmt2p complex in this respect is unclear. Here, we have identified the binding partners of yeast Pmt1p and Pmt2p. These include ER chaperones involved in oxidative protein folding; the Hrd1p complex, which is involved in ER-associated protein degradation (ERAD); and the p24 protein complex involved in ER export. The results suggest that the Pmt1p-Pmt2p complex participates in these processes. We tested this assumption in a functional assay and found that whereas the Pmt1p-Pmt2p complex promotes fast ER export of the GPI-anchored protein Gas1p, it retains the misfolded version Gas1*p and targets it to the Hrd1p complex for subsequent degradation. Our results reveal previously unknown cellular roles of the Pmt1p-Pmt2p complex in connection with the ERAD machinery and show its participation in ER protein quality control.Ministerio de Ciencia BFU2009-0729

Competition between neighbouring topogenic signals during membrane protein insertion into the ER membrane

To better define the mechanism of membrane protein insertion into the membrane of the endoplasmic reticulum, we measured the kinetics of translocation across microsomal membranes of the N-terminal lumenal tail and the lumenal domain following the second transmembrane segment (TM2) in the multispanning mouse protein Cig30. In the wild-type protein, the N-terminal tail translocates across the membrane before the downstream lumenal domain. Addition of positively charged residues to the N-terminal tail dramatically slows down its translocation and allows the downstream lumenal domain to translocate at the same time as or even before the N-tail. When TM2 is deleted, or when the loop between TM1 and TM2 is lengthened, addition of positively charged residues to the N-terminal tail causes TM1 to adopt an orientation with its N-terminal end in the cytoplasm. We suggest that the topology of the TM1-TM2 region of Cig30 depends on a competition between TM1 and TM2 such that the transmembrane segment that inserts first into the ER membrane determines the final topology

Study of polytopic membrane protein topological organization as a function of membrane lipid composition.

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM). SCAM is adapted to follow changes in membrane protein topology as a function of changes in membrane lipid composition. The strategy described can be adapted to any membrane system

Distant downstream sequence determinants can control N-tail translocation during protein insertion into the Endoplasmic Reticulum membrane

We have studied the membrane insertion of ProW, an Escherichia coli inner membrane protein with seven transmembrane segments and a large periplasmic N-terminal tail, into endoplasmic reticulum (ER)-derived dog pancreas microsomes. Strikingly, significant levels of N-tail translocation is seen only when a minimum of four of the transmembrane segments are present; for constructs with fewer transmembrane segments, the N-tail remains mostly nontranslocated and the majority of the molecules adopt an 'inverted' topology where normally nontranslocated parts are translocated and vice versa. N-tail translocation can also be promoted by shortening of the N-tail and by the addition of positively charged residues immediately downstream of the first trasnmembrane segment. We conclude that as many as four consecutive transmembrane segments may be collectively involved in determining membrane protein topology in the ER and that the effects of downstream sequence determinants may vary depending on the size and charge of the N-tail. We also provide evidence to suggest that the ProW N-tail is translocated across the ER membrane in a C-to-N-terminal direction

A unifying mechanism for the biogenesis of membrane proteins co-operatively integrated by the Sec and Tat pathways

The majority of multi-spanning membrane proteins are co-translationally inserted into the bilayer by the Sec pathway. An important subset of membrane proteins have globular, cofactor-containing extracytoplasmic domains requiring the dual action of the co-translational Sec and post-translational Tat pathways for integration. Here, we identify further unexplored families of membrane proteins that are dual Sec-Tat-targeted. We establish that a predicted heme-molybdenum cofactor-containing protein, and a complex polyferredoxin, each require the concerted action of two translocases for their assembly. We determine that the mechanism of handover from Sec to Tat pathway requires the relatively low hydrophobicity of the Tat-dependent transmembrane domain. This, coupled with the presence of C-terminal positive charges, results in abortive insertion of this transmembrane domain by the Sec pathway and its subsequent release at the cytoplasmic side of the membrane. Together, our data points to a simple unifying mechanism governing the assembly of dual targeted membrane proteins.</jats:p

A survey on feature weighting based K-Means algorithms

This is a pre-copyedited, author-produced PDF of an article accepted for publication in Journal of Classification [de Amorim, R. C., 'A survey on feature weighting based K-Means algorithms', Journal of Classification, Vol. 33(2): 210-242, August 25, 2016]. Subject to embargo. Embargo end date: 25 August 2017. The final publication is available at Springer via http://dx.doi.org/10.1007/s00357-016-9208-4 © Classification Society of North America 2016In a real-world data set there is always the possibility, rather high in our opinion, that different features may have different degrees of relevance. Most machine learning algorithms deal with this fact by either selecting or deselecting features in the data preprocessing phase. However, we maintain that even among relevant features there may be different degrees of relevance, and this should be taken into account during the clustering process. With over 50 years of history, K-Means is arguably the most popular partitional clustering algorithm there is. The first K-Means based clustering algorithm to compute feature weights was designed just over 30 years ago. Various such algorithms have been designed since but there has not been, to our knowledge, a survey integrating empirical evidence of cluster recovery ability, common flaws, and possible directions for future research. This paper elaborates on the concept of feature weighting and addresses these issues by critically analysing some of the most popular, or innovative, feature weighting mechanisms based in K-Means.Peer reviewedFinal Accepted Versio