Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

被引:38
作者
Kida, Yuichiro [1 ,2 ]
Morimoto, Fumiko [1 ]
Sakaguchi, Masao [1 ,2 ]
机构
[1] Univ Hyogo, Grad Sch Life Sci, Kobe, Hyogo 6781297, Japan
[2] Japan Sci & Technol Agcy, CREST, Kobe, Hyogo 6781297, Japan
关键词
D O I
10.1083/jcb.200707050
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61 alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.
引用
收藏
页码:1441 / 1452
页数:12
相关论文
共 46 条
[21]   Reorientation of aquaporin-1 topology during maturation in the endoplasmic reticulum [J].
Lu, Y ;
Turnbull, IR ;
Bragin, A ;
Carveth, K ;
Verkman, AS ;
Skach, WR .
MOLECULAR BIOLOGY OF THE CELL, 2000, 11 (09) :2973-2985
[22]   SecYEG assembles into a tetramer to form the active protein translocation channel [J].
Manting, EH ;
van der Does, C ;
Remigy, H ;
Engel, A ;
Driessen, AJM .
EMBO JOURNAL, 2000, 19 (05) :852-861
[23]   Architecture of the ribosome-channel complex derived from native membranes [J].
Ménétret, JF ;
Hegde, RS ;
Heinrich, SU ;
Chandramouli, P ;
Ludtke, SJ ;
Rapoport, TA ;
Akey, CW .
JOURNAL OF MOLECULAR BIOLOGY, 2005, 348 (02) :445-457
[24]   Structure of the E-coli protein-conducting channel bound to a translating ribosome [J].
Mitra, K ;
Schaffitzel, C ;
Shaikh, T ;
Tama, F ;
Jenni, S ;
Brooks, CL ;
Ban, N ;
Frank, J .
NATURE, 2005, 438 (7066) :318-324
[25]   NHE6 protein possesses a signal peptide destined for endoplasmic reticulum membrane and localizes in secretory organelles of the cell [J].
Miyazaki, E ;
Sakaguchi, M ;
Wakabayashi, S ;
Shigekawa, M ;
Mihara, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (52) :49221-49227
[26]   Structure of the mammalian ribosome-channel complex at 17 Å resolution [J].
Morgan, DG ;
Ménétret, JF ;
Neuhof, A ;
Rapoport, TA ;
Akey, CW .
JOURNAL OF MOLECULAR BIOLOGY, 2002, 324 (04) :871-886
[27]   Fluorescence resonance energy transfer analysis of protein translocase -: SecYE from Thermus thermophilus HB8 forms a constitutive oligomer in membranes [J].
Mori, H ;
Tsukazaki, T ;
Masui, R ;
Kuramitsu, S ;
Yokoyama, S ;
Johnson, AE ;
Kimura, Y ;
Akiyama, Y ;
Ito, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (16) :14257-14264
[28]   Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel [J].
Osborne, Andrew R. ;
Rapoport, Tom A. .
CELL, 2007, 129 (01) :97-110
[29]   Protein translocation by the Sec61/SecY channel [J].
Osborne, AR ;
Rapoport, TA ;
van den Berg, B .
ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, 2005, 21 :529-550
[30]   Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence [J].
Ota, K ;
Sakaguchi, M ;
Hamasaki, N ;
Mihara, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (38) :29743-29748