scholarly journals Identifying Interacting Proteins of Arabidopsis Cyclophilin38 (AtCYP38), a Key Factor for PSII Assembly/Repair, via Multiple Screening Approaches

2020 ◽  
Author(s):  
Yaqi Hao ◽  
Jiashu Chu ◽  
Lujing Shi ◽  
Cong Ma ◽  
Liangliang Hui ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Thylakoid Membrane ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Working Mechanism ◽  
Web Based ◽  
Thylakoid Lumen ◽  
Target Proteins ◽  
Thylakoid Membrane Proteins ◽  
Two Hybrid

Abstract BackgroundAtCYP38, a thylakoid lumen localized immunophilin, is essential for photosystem II (PSII) assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen to influence PSII performance. Therefore, identifying its target proteins and cofactors would be a key step to understand the working mechanism of AtCYP38.ResultsTo identify potential interacting proteins of AtCYP38, we first adopted two web-based tools, ATTED-II and STRING, and found 15 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with the N-terminal domain, the C-terminal domain, and the full-length mature protein of AtCYP38. 25 positive targets were identified, but a very limited number of target proteins were localized in the thylakoid lumen. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we created a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumen fractions of thylakoid membrane proteins. After screening the mini library with 3 different forms of AtCYP38, we obtained 6 thylakoid membrane proteins and 9 thylakoid lumenal proteins as interacting proteins of AtCYP38. We further confirmed the localization of several identified proteins and their interaction between AtCYP38.ConclusionsAfter analysis with two web-based tools and yeast two-hybrid screenings against two different libraries, we identified a couple of potential interacting proteins, which could be functionally related to AtCYP38. We believe that the results will lay a foundation for unveiling the working mechanism of AtCYP38 in photosynthesis.

Characterisation of Zea mays L. plastidial transglutaminase: interactions with thylakoid membrane proteins

Plant Biology ◽  
2010 ◽  
Vol 12 (5) ◽  
pp. 708-716 ◽  
Author(s):  
A. Campos ◽  
P. K. Carvajal-Vallejos ◽  
E. Villalobos ◽  
C. F. Franco ◽  
A. M. Almeida ◽  
...  
Keyword(s):  
Zea Mays ◽  
Membrane Proteins ◽  
Thylakoid Membrane ◽  
Zea Mays L ◽  
Thylakoid Membrane Proteins

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

scholarly journals The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane

2000 ◽  
Vol 149 (6) ◽  
pp. 1171-1178 ◽  
Author(s):  
William B. Snyder ◽  
Antonius Koller ◽  
A. Jobu Choy ◽  
Suresh Subramani
Keyword(s):  
Membrane Proteins ◽  
Multiple Integral ◽  
Integral Membrane Proteins ◽  
Peroxisome Biogenesis ◽  
Yeast Two Hybrid ◽  
Peroxisomal Membrane ◽  
Site Of Action ◽  
Peroxisome Membrane ◽  
Two Hybrid ◽  
New Protein

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

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