Glycosylphosphatidylinositol (GPI)-linked aspartyl proteases regulate vacuole homeostasis in Candida glabrata
A family of eleven glycosylphosphatidylinositol (GPI)-linked, cell surface-associated aspartyl proteases (yapsins)in a human opportunistic fungal pathogen Candida glabrata is required for cell wall remodeling and pH homeostasis, survival in macrophages and virulence in a murine model of disseminated candidiasis. Here, we report new roles for yapsins in C. glabrata physiology and implicate them for the first time in the regulation of vacuole homeostasis. We show that a C. glabrata mutant lacking all eleven yapsins, Cgyps1-11∆, possesses an enlarged vacuole and displays vma- (vacuolar membrane ATPase)-like phenotypes ...
Source: BJ Cell - December 17, 2013 Category: Biochemistry Authors: G Bairwa, M Rasheed, R Taigwal, R Sahoo, R Kaur Tags: BJ Cell Source Type: research
Distinct regions of triadin are required for targeting and retention at the junctional domain of the sarcoplasmic reticulum
Ca2+ release necessary for muscle contraction occurs at the junctional domain of the sarcoplasmic reticulum (j-SR). It requires the assembly of a large multi-protein complex containing the ryanodine receptor (RyR) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multi-protein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1, TR2 and TR3, that contribute to the localization of...
Source: BJ Cell - December 11, 2013 Category: Biochemistry Authors: D Rossi, C Bencini, M Maritati, F Benini, S Lorenzini, E Pierantozzi, A Maria Scarcella, C Paolini, F Protasi, V Sorrentino Tags: BJ Cell Source Type: research
The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3; 3b, 3i and 3h
Eukaryotic initiation factor 3 (eIF3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals eIF3 is composed of thirteen different polypeptide subunits five of which, a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. Here we show that the eukaryotic cytosolic chaperonin CCT binds to newly synthesised eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, over-expression of the latter two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of g...
Source: BJ Cell - December 10, 2013 Category: Biochemistry Authors: A Roobol, J Roobol, M J Carden, M E Smith, J WB Hershey, A Bastide, J RP Knight, A E Willis, C Mark Smales Tags: BJ Cell Source Type: research
Dynamics of mitochondrial Ca2{+} uptake in MICU1-knockdown cells
MICU1 is an important regulator of the mitochondrial Ca2+-uniporter (MCU) that has been recently shown to act as a gatekeeper of MCU at low cytosolic [Ca2+] ([Ca2+]c). We have studied here in detail the dynamics of MCU activity after shRNA-knockdown of MICU1 and we find several new interesting properties. In MICU1-knockdown cells, the rate of mitochondrial Ca2+-uptake was largely increased at low [Ca2+]c (<2µM), but it was decreased at high [Ca2+]c (>4µM). In the 2-4µM range, a mixed behavior was observed, where mitochondrial Ca2+-uptake started ea...
Source: BJ Cell - December 9, 2013 Category: Biochemistry Authors: S de la Fuente, J Matesanz-Isabel, R I. Fonteriz, M Montero, J Alvarez Tags: BJ Cell Source Type: research
Transmembrane Serine of Rot1 protein is essential for yeast cell viability
Polar residues are present in transmembrane (TM) helices and may influence the folding or association of membrane proteins. Here, we use an in vivo approach to analyse the functional or structural role of amino acid residues in membrane-spanning motifs using Rot1 protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and contains a single TM domain. An Ala-insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical Ser residue inside the helix that plays an essential role in maintaining cell viabili...
Source: BJ Cell - December 5, 2013 Category: Biochemistry Authors: C A. Martínez-Garay, M Juanes, J Igual, I Mingarro, M Bañó Tags: BJ Biomolecules Source Type: research
Dynamics of G{alpha}q-protein-p63RhoGEF interaction and its regulation by RGS2
Some G‑protein‑coupled receptors regulate biological processes via Gα12/13‑ or Gαq/11‑mediated stimulation of Rho guanine nucleotide exchange factors (RhoGEFs). p63RhoGEF is known to be specifically activated by Gαq/11 and mediates a major part of the acute response of vascular smooth muscle cells to Angiotensin II treatment. In order to gain information about the dynamics of receptor-mediated activation of p63RhoGEF we developed a FRET-based assay to study interactions between Gαq-CFP and Venus-p63RhoGEF in single living cells. Upon activation of histaminergic H1 or ...
Source: BJ Cell - December 4, 2013 Category: Biochemistry Authors: E Bodmann, A Rinne, D Brandt, S Lutz, T Wieland, R Grosse, M Bünemann Tags: BJ Signal Source Type: research
The Escherichia coli RNA processing and degradation machinery is compartmentalized within an organized cellular network
Bacterial RNA processing and degradation involves the coordinated action of a large number of RNases, RNA helicases and other proteins. It is not known how this functional network is organized within the cell nor how it is coordinated or regulated. Here we show that multiple components of the RNA degradation and processing network of Escherichia coli are localized within extended cellular structures that appear to coil around the periphery of the cell. These include Orn, Hfq, PAP I, RNase III, RppH, RraA and RraB in addition to the previously reported proteins RNase II and RNaseE. Double label localization studies of sever...
Source: BJ Cell - November 25, 2013 Category: Biochemistry Authors: A Taghbalout, Q Yang, V Arluison Tags: BJ Gene Source Type: research
Arl6IP1 has the potency to shape the mammalian ER membrane in a reticulon-like fashion
The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of membrane sheets and tubules. Two classes of the evolutionally conserved ER membrane proteins, reticulons and REEPs/DP1/Yop1, shape high-curvature ER tubules. In mammals, four members of the reticulon family and six members of the REEP family have been identified so far. Here we report that ADP-ribosylation factor-like 6 interacting protein 1 (Arl6IP1), an anti-apoptotic protein specific to multicellular organisms, is a potential player in shaping the ER tubules in mammalian cells. Arl6IP1, which does not share overall primary sequen...
Source: BJ Cell - November 22, 2013 Category: Biochemistry Authors: Y Yamamoto, A Yoshida, N Miyazaki, K Iwasaki, T Sakisaka Tags: BJ Cell Source Type: research
The connexin43-interacting protein of 75kDa (CIP75) mediates the endoplasmic reticulum dislocation of connexin43
Gap junctions are intercellular channels comprised of connexin proteins, such as connexin43 (Cx43). The level of gap junctional intercellular communication can be regulated by Cx43 turnover mediated through various degradation pathways. The UbL (ubiquitin-like domain)-UBA (ubiquitin-associated domain) protein, CIP75, regulates the proteasomal degradation of Cx43. Subcellular fractionation studies indicated that CIP75 interacts with Cx43 that is localized to the membrane of the endoplasmic reticulum (ER). This Cx43-CIP75 complex also contained the proteasomal subunits S2/Rpn1 and S5a/Rpn10, as demonstrated by co-immunopreci...
Source: BJ Cell - November 20, 2013 Category: Biochemistry Authors: V Su, C Hoang, D Geerts, A F. Lau Tags: BJ Cell Source Type: research
Myogenic bladder defects in mouse models of human oculodentodigital dysplasia
To date, over 65 mutations in the gene encoding Cx43 have been linked to the autosomal dominant disease, oculodentodigital dysplasia (ODDD). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. Bladder smooth muscle cells (BSMCs) from wild-type and two Cx43 mutant lines (Cx43G60s and Cx43I130T) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs fro...
Source: BJ Cell - November 15, 2013 Category: Biochemistry Authors: T Huang, Q Shao, K Barr, J Simek, G I. Fishman, D W. Laird Tags: BJ Disease Source Type: research
Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor
In this study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR’s interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR and its interaction with the SUG1 subunit of the 26S proteasome. Together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity. (Source: BJ Cell)
Source: BJ Cell - November 14, 2013 Category: Biochemistry Authors: T Chen, E M. Laurenzana, D M. Coslo, F Chen, C J. Omiecinski Tags: BJ Cell Source Type: research
SIM-dependent enhancement of substrate-specific sumoylation by a ubiquitin ligase in vitro
SUMO interaction motifs (SIMs), which mediate the non-covalent binding of the small ubiquitin-related modifier (SUMO) to other proteins, are usually involved in the recognition of sumoylated substrates by downstream effectors that transmit the biological signal of the modification. In budding yeast ubiquitin ligase Rad18, a SIM contributes to the recognition of sumoylated PCNA as its physiological ubiquitylation target. We now show that Rad18 is also capable of enhancing PCNA sumoylation in a SIM-dependent manner in vitro, most likely by means of directing SUMO-loaded Ubc9 towards the substrate. The process shares importan...
Source: BJ Cell - November 14, 2013 Category: Biochemistry Authors: J L. Parker, H D. Ulrich Tags: BJ Signal Source Type: research
Four-and-a-half LIM domains proteins are novel regulators of the protein kinase D pathway in cardiac myocytes
Protein kinase D (PKD) is a Ser/Thr kinase implicated in multiple cardiac roles, including the phosphorylation of the class II histone deacetylase HDAC5 and thereby de-repression of myocyte enhancer factor 2 (MEF2) transcription factor activity. Here we identify four-and-a-half LIM domains proteins 1 and 2 (FHL1 and FHL2) as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult or neonatal rat ventricular myocytes (NRVM), respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PK...
Source: BJ Cell - November 13, 2013 Category: Biochemistry Authors: K Stathopoulou, F Cuello, A J. Candasamy, E M. Kemp, E Ehler, R S. Haworth, M Avkiran Tags: BJ Signal Source Type: research
Membrane lipids are key-modulators of the endocannabinoid-hydrolase FAAH
We report that FAAH dimer is stabilized by the lipid bilayer and shows higher membrane binding affinity and enzymatic activity within membranes containing both cholesterol and the natural FAAH substrate, anandamide (AEA). Additionally, colocalization of cholesterol, AEA, and FAAH in mouse neuroblastoma cells suggests a mechanism through which cholesterol increases the substrate accessibility of FAAH. (Source: BJ Cell)
Source: BJ Cell - November 12, 2013 Category: Biochemistry Authors: E Dainese, G De Fabritiis, A Sabatucci, S Oddi, C Beatrice Angelucci, C Di Pancrazio, T Giorgino, N Stanley, M Del Carlo, B Cravatt, M Maccarrone Tags: BJ Biomolecules Source Type: research
The role of EDEM2 versus EDEM1 in ricin transport from the endoplasmic reticulum to the cytosol
In this study we report that EDEM2 is also involved in ricin retrotranslocation out of the ER. However, the role of EDEM1 and EDEM2 in ricin transport to the cytosol seems to differ. EDEM2 promotes ricin retrotranslocation irrespectively of ER translocon accessibility; moreover co- immunoprecipitation and pull-down studies revealed that more ricin can interact with EDEM2 in comparison to EDEM1. On the other hand, interactions of both lectins with RTA are dependent on the structure of ricin A-chain. Thus, our data display a newly discovered role for EDEM2. Moreover, analysis of the involvement of EDEM1 and EDEM2 in ricin re...
Source: BJ Cell - November 8, 2013 Category: Biochemistry Authors: M Słomińska-Wojewódzka, A Pawlik, I Sokołowska, J Antoniewicz, G Węgrzyn, K Sandvig Tags: BJ Cell Source Type: research
