O-Mannosylation is required for the solubilization of heterologously expressed human beta-amyloid precursor protein in Saccharomyces cerevisiae.
In an attempt to express human beta-amyloid precursor protein (APP) in yeast, we fortuitously found that this protein is only O-glycosylated in yeast. APP was effectively expressed in yeast, processed by yeast alpha-secretases, members of the Yapsin family, to produce N-terminal (sAPPalpha) and C-terminal (CTFalpha) domains, when its signal sequence was replaced by that of the yeast alpha-mating factor. APP is known to acquire N- and O-glycosylation through the endoplasmic reticulum (ER) and the Golgi apparatus and is transported to the plasma membrane in mammalian cells. In spite of the presence of canonical N-glycosylation consensus sequences, APP was not N-glycosylated in the yeast system. Pulse-chase experiments demonstrated that APP received only O-mannosylation in yeast. Examination of yeast pmt mutants, which are defective in the initiation of O-mannosylation in the ER, revealed that Pmt4p is most responsible for the oligosaccharide modification of APP. Maturation of APP was slowed down and aggregated forms of APP were observed by sucrose density gradient fractionation of the Deltapmt4 mutant lysate. This caused decreased production of CTFalpha. We conclude that O-mannosylation is required for the solubilization of exogenously expressed human APP.
Murakami-Sekimata
Akiko
A
RIKEN Brain Science Institute, Wako, Saitama, Japan.
Sato
Ken
K
Sato
Ken
K
Takashima
Akihiko
A
Nakano
Akihiko
A
D016564:Amyloid beta-Protein Precursor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015966:Gene Expression Regulation, Fungal; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D011497:Protein O-Methyltransferase / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D011994:Recombinant Proteins; D011497:Protein O-Methyltransferase; D008358:Mannose