Membrane translocation of lumenal domains of membrane proteins powered by downstream transmembrane sequences.
Translocation of the N-terminus of a type I signal anchor (SA-I) sequence across the endoplasmic reticulum membrane can be arrested by tagging with a streptavidin-binding peptide tag (SBP tag) and trapping by streptavidin. In the present study, we first examine the affinity required for the translocation arrest. When the SBP tag is serially truncated, the ability for arrest gradually decreases. Surface plasmon resonance analysis shows that an interaction as strong as 10(-8) M or a smaller dissociation constant is required for trapping the topogenesis of a natural SA-I sequence. Such truncated tags, however, become effective by mutating the SA-I sequence, suggesting that the translocation motivation is considerably influenced by the properties of the SA-I sequence. In addition, we introduce the SBP tag into lumenal loops of a multispanning membrane protein, human erythrocyte band 3. Among the tagged loops between transmembrane 1 (TM1) and TM8, three loops are trapped by cytosolic streptavidin. These loops are followed by TM sequences possessing topogenic properties, like the SA-I sequence, and translocation of one loop is diminished by insertion of a proline into the following TM sequence. These findings suggest that the translocation of lumenal loops by SA-I-like TM sequences has a crucial role in topogenesis of multispanning membrane proteins.
Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan.
Takaaki
T
Yabuki
Fumiko
F
Morimoto
Yuichiro
Y
Kida
Masao
M
Sakaguchi
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics*; D019809:Streptavidin
D002352:Carrier Proteins; D008565:Membrane Proteins; C452700:streptavidin-binding peptide; D019809:Streptavidin