Site-specific photocross-linking reveals that Sec61p and TRAM contact different regions of a membrane-inserted signal sequence.
A chemically charged amber suppressor tRNA was used to introduce the photoactivatable amino acid (Tmd)Phe at a selected position within the signal sequence of the secretory protein preprolactin. This allowed the interactions of the NH2-terminal, the central, and the COOH-terminal regions of the signal sequence to be investigated during insertion into the membrane of the endoplasmic reticulum (ER). We found that different regions of the nascent chains were photocross-linked to different ER proteins. The TRAM protein (translocating chain-associating membrane protein) contacts the NH2-terminal region of the signal sequence while the mammalian Sec61p contacts the hydrophobic core of the signal sequence and regions COOH-terminal of this. These results suggest that the ER translocation complex is composed of heterologous protein subunits which contact distinct regions of nascent polypeptides during their membrane insertion.
High
S
S
European Molecular Biology Laboratory, Heidelberg, Germany.
Martoglio
B
B
Görlich
D
D
Andersen
S S
SS
Ashford
A J
AJ
Giner
A
A
Hartmann
E
E
Prehn
S
S
Rapoport
T A
TA
Dobberstein
B
B
D000215:Acylation; D000818:Animals; D003062:Codon; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry; D000069816:SEC Translocation Channels
D003062:Codon; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; C074282:translocating chain-associating membrane protein (TRAM); D012343:RNA, Transfer