TY - JOUR
T1 - Novel interactome of Saccharomyces cerevisiae myosin type II identified by a modified integrated membrane yeast two-hybrid (iMYTH) screen
AU - Santiago, Ednalise
AU - Akamine, Pearl
AU - Snider, Jamie
AU - Wong, Victoria
AU - Jessulat, Matthew
AU - Deineko, Viktor
AU - Gagarinova, Alla
AU - Aoki, Hiroyuki
AU - Minic, Zoran
AU - Phanse, Sadhna
AU - Antonio, Andrea San
AU - Cubano, Luis A.
AU - Rymond, Brian C.
AU - Babu, Mohan
AU - Stagljar, Igor
AU - Rodriguez-Medina, Jose R.
N1 - Publisher Copyright:
© 2016 Santiago et al.
PY - 2016/5/1
Y1 - 2016/5/1
N2 - Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.
AB - Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other functions of the Myo1p tail domain. To identify novel Myo1p-interacting proteins, and determine if Myo1p can serve as a scaffold to recruit proteins to the bud neck during cytokinesis, we used the integrated split-ubiquitin membrane yeast two-hybrid (iMYTH) system. Myo1p was iMYTH-tagged at its C-terminus, and screened against both cDNA and genomic prey libraries to identify interacting proteins. Control experiments showed that the Myo1p-bait construct was appropriately expressed, and that the protein colocalized to the yeast bud neck. Thirty novel Myo1p-interacting proteins were identified by iMYTH. Eight proteins were confirmed by coprecipitation (Ape2, Bzz1, Fba1, Pdi1, Rpl5, Tah11, and Trx2) or mass spectrometry (AP-MS) (Abp1). The novel Myo1p-interacting proteins identified come from a range of different processes, including cellular organization and protein synthesis. Actin assembly/disassembly factors such as the SH3 domain protein Bzz1 and the actin-binding protein Abp1 represent likely Myo1p interactions during cytokinesis.
KW - Cytokinesis
KW - Interactome
KW - Myo1p
KW - Proteomics
KW - Yeast
UR - http://www.scopus.com/inward/record.url?scp=84966389661&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84966389661&partnerID=8YFLogxK
U2 - 10.1534/g3.115.026609
DO - 10.1534/g3.115.026609
M3 - Article
C2 - 26921299
AN - SCOPUS:84966389661
SN - 2160-1836
VL - 6
SP - 1469
EP - 1474
JO - G3: Genes, Genomes, Genetics
JF - G3: Genes, Genomes, Genetics
IS - 5
ER -