scholarly journals Structural determinants of the specificity of a membrane binding domain of the scaffold protein Ste5 of budding yeast: Implications in signaling by the scaffold protein in MAPK pathway

2012 ◽  
Vol 1818 (5) ◽  
pp. 1250-1260 ◽  
Author(s):  
Anirban Bhunia ◽  
Harini Mohanram ◽  
Surajit Bhattacharjya
Keyword(s):  
Budding Yeast ◽  
Mapk Pathway ◽  
Membrane Binding ◽  
Scaffold Protein ◽  
Binding Domain ◽  
Structural Determinants

scholarly journals Structure of a Membrane-binding Domain from a Non-enveloped Animal Virus

2006 ◽  
Vol 281 (39) ◽  
pp. 29278-29286 ◽  
Author(s):  
Lenize F. Maia ◽  
Márcia R. Soares ◽  
Ana P. Valente ◽  
Fabio C. L. Almeida ◽  
Andréa C. Oliveira ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Binding Domain ◽  
Animal Virus

scholarly journals A Membrane Binding Domain in the Ste5 Scaffold Synergizes with Gβγ Binding to Control Localization and Signaling in Pheromone Response

2005 ◽  
Vol 20 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Matthew J. Winters ◽  
Rachel E. Lamson ◽  
Hideki Nakanishi ◽  
Aaron M. Neiman ◽  
Peter M. Pryciak
Keyword(s):  
Membrane Binding ◽  
Binding Domain ◽  
Pheromone Response

scholarly journals Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Richard Izrael ◽  
Lívia Marton ◽  
Gergely N. Nagy ◽  
Hajnalka L. Pálinkás ◽  
Nóra Kucsma ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cell Line ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Membrane Binding ◽  
Binding Domain ◽  
Regulatory Function ◽  
Phosphocholine Cytidylyltransferase ◽  
Phosphatidylcholine Biosynthesis ◽  
Localization Signal

AbstractThe phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

scholarly journals Structural determinants for protein unfolding and translocation by the Hsp104 protein disaggregase

2017 ◽  
Vol 37 (6) ◽  
Author(s):  
Jungsoon Lee ◽  
Nuri Sung ◽  
Lythou Yeo ◽  
Changsoo Chang ◽  
Sukyeong Lee ◽  
...  
Keyword(s):  
Protein Unfolding ◽  
Substrate Binding ◽  
Binding Domain ◽  
Atp Binding ◽  
Structural Determinants ◽  
Functional Studies ◽  
Loop 2 ◽  
Loop Conformation ◽  
Do So

The ring-forming Hsp104 ATPase cooperates with Hsp70 and Hsp40 molecular chaperones to rescue stress-damaged proteins from both amorphous and amyloid-forming aggregates. The ability to do so relies upon pore loops present in the first ATP-binding domain (AAA-1; loop-1 and loop-2 ) and in the second ATP-binding domain (AAA-2; loop-3) of Hsp104, which face the protein translocating channel and couple ATP-driven changes in pore loop conformation to substrate translocation. A hallmark of loop-1 and loop-3 is an invariable and mutational sensitive aromatic amino acid (Tyr257 and Tyr662) involved in substrate binding. However, the role of conserved aliphatic residues (Lys256, Lys258, and Val663) flanking the pore loop tyrosines, and the function of loop-2 in protein disaggregation has not been investigated. Here we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 exhibiting molecular interactions involving both AAA-1 pore loops, which resemble contacts with bound substrate. Corroborated by biochemical experiments and functional studies in yeast, we show that aliphatic residues flanking Tyr257 and Tyr662 are equally important for substrate interaction, and abolish Hsp104 function when mutated to glycine. Unexpectedly, we find that loop-2 is sensitive to aspartate substitutions that impair Hsp104 function and abolish protein disaggregation when loop-2 is replaced by four aspartate residues. Our observations suggest that Hsp104 pore loops have non-overlapping functions in protein disaggregation and together coordinate substrate binding, unfolding, and translocation through the Hsp104 hexamer.

scholarly journals Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

1997 ◽  
Vol 325 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Jilin YANG ◽  
Jinxia WANG ◽  
Irene TSEU ◽  
Maciej KULISZEWSKI ◽  
Wensu LEE ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Fusion Proteins ◽  
Membrane Binding ◽  
Lipid Vesicles ◽  
Lipid Binding ◽  
Binding Domain ◽  
Membrane Interactions ◽  
Phosphocholine Cytidylyltransferase ◽  
N Terminus ◽  
L2 Cells

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.

Fluorescence study of a mutant cytochrome b5 with a single tryptophan in the membrane-binding domain

Biochemistry ◽  
1991 ◽  
Vol 30 (42) ◽  
pp. 10200-10206 ◽  
Author(s):  
Alexey S. Ladokhin ◽  
L. Wang ◽  
A. W. Steggles ◽  
Peter W. Holloway
Keyword(s):  
Cytochrome B5 ◽  
Membrane Binding ◽  
Binding Domain ◽  
Fluorescence Study

scholarly journals The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition

2015 ◽  
Vol 211 (2) ◽  
pp. 309-322 ◽  
Author(s):  
Lindsay G. Lammers ◽  
Steven M. Markus
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Coiled Coil ◽  
Binding Domain ◽  
Cell Cortex ◽  
Wild Type ◽  
Microtubule Binding ◽  
Coiled Coil Domain ◽  
Microtubule Binding Domain

Cortically anchored dynein orients the spindle through interactions with astral microtubules. In budding yeast, dynein is offloaded to Num1 receptors from microtubule plus ends. Rather than walking toward minus ends, dynein remains associated with plus ends due in part to its association with Pac1/LIS1, an inhibitor of dynein motility. The mechanism by which dynein is switched from “off” at the plus ends to “on” at the cell cortex remains unknown. Here, we show that overexpression of the coiled-coil domain of Num1 specifically depletes dynein–dynactin–Pac1/LIS1 complexes from microtubule plus ends and reduces dynein-Pac1/LIS1 colocalization. Depletion of dynein from plus ends requires its microtubule-binding domain, suggesting that motility is required. An enhanced Pac1/LIS1 affinity mutant of dynein or overexpression of Pac1/LIS1 rescues dynein plus end depletion. Live-cell imaging reveals minus end–directed dynein–dynactin motility along microtubules upon overexpression of the coiled-coil domain of Num1, an event that is not observed in wild-type cells. Our findings indicate that dynein activity is directly switched “on” by Num1, which induces Pac1/LIS1 removal.

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