TY - JOUR
T1 - Mutations in S-adenosylhomocysteine hydrolase (AHCY) affect its nucleocytoplasmic distribution and capability to interact with S-adenosylhomocysteine hydrolase-like 1 protein
AU - Grbeša, Ivana
AU - Kalo, Alon
AU - Belužić, Robert
AU - Kovačević, Lucija
AU - Lepur, Adriana
AU - Rokić, Filip
AU - Hochberg, Hodaya
AU - Kanter, Itamar
AU - Simunović, Vesna
AU - Muńoz-Torres, Pau Marc
AU - Shav-Tal, Yaron
AU - Vugrek, Oliver
N1 - Publisher Copyright:
© 2017 The Authors
PY - 2017/9
Y1 - 2017/9
N2 - S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.
AB - S-adenosylhomocysteine hydrolase (AHCY) is thought to be located at the sites of ongoing AdoMet-dependent methylation, presumably in the cell nucleus. Endogenous AHCY is located both in cytoplasm and the nucleus. Little is known regarding mechanisms that drive its subcellular distribution, and even less is known on how mutations causing AHCY deficiency affect its intracellular dynamics. Using fluorescence microscopy and GFP-tagged AHCY constructs we show significant differences in the intensity ratio between nuclei and cytoplasm for mutant proteins when compared with wild type AHCY. Interestingly, nuclear export of AHCY is not affected by leptomycin B. Systematic deletions showed that AHCY has two regions, located at both sides of the protein, that contribute to its nuclear localization, implying the interaction with various proteins. In order to evaluate protein interactions in vivo we engaged in bimolecular fluorescence complementation (BiFC) based studies. We investigated previously assumed interaction with AHCY-like-1 protein (AHCYL1), a paralog of AHCY. Indeed, significant interaction between both proteins exists. Additionally, silencing AHCYL1 leads to moderate inhibition of nuclear export of endogenous AHCY.
KW - AHCY
KW - AHCYL1 (IRBIT)
KW - BiFC
KW - LMB1
KW - NES
KW - Nuclear export
UR - http://www.scopus.com/inward/record.url?scp=85021119352&partnerID=8YFLogxK
U2 - 10.1016/j.ejcb.2017.05.002
DO - 10.1016/j.ejcb.2017.05.002
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C2 - 28647132
SN - 0171-9335
VL - 96
SP - 579
EP - 590
JO - European Journal of Cell Biology
JF - European Journal of Cell Biology
IS - 6
ER -