MOLECULAR CELL BIOLOGY SIXTH EDITION MOLECULAR CELL BIOLOGY SIXTH EDITION Copyright 2008 © W. H. Freeman and Company CHAPTER 13 Moving Proteins into Membranes and Organelles CHAPTER 13 Moving Proteins into Membranes and Organelles Lodish Berk Kaiser Krieger Scott Bretscher Ploegh Matsudaira © 2008 W. H. Freeman and Company

Secretory pathway Cytosol Ribosomes ER ribosomes ER signal peptide Conformation Signal sequences (signal peptide) Translocation channel Usually requires energy Signal peptide removed before maturation

ER Secretory Pathway Protein synthesis and translocation into the ER Protein folding and modification Protein transport to Gogi, lysosomes or cell surface using vesicles. N-terminus peptide sequence of 16 to 30 a.a. 1 or more positively charge a.a. follow by a stretch of 6-12 hydrophobic residues. Is cleaved before translation is completed.

Cotranslational translocation

SRP: signal recognition particle Cytsolic ribonucleoprotein particle Binds to TP and ribosome, and SRP receptor SRP inhibits translation if microsomes are absent If protein is too long it can’t be translocated Translocon: protein channel Protein is not folded until completely in the ER lumen

No ATP require just the push from the elongated peptide. Signal peptidase: cut the signal peptide BiP molecular chaperone also help in moving the protein to the ER lumen and help in the proper folding of the polypeptide.

Topogenic sequences: direct membrane integration and orientation

Type I: Hydrophobic stop-transfer anchor sequence

Type II: Signal anchor sequence and is oriented by positively charged a.a. in the N-terminal of SAS

Type III: Signal anchor sequence and is oriented by positively charged a.a. in the C-terminal of SAS

Type V: Glycosylphosphatidylinosotol (GPI)

Type V: Use of a transamidase

MT: amphipathic N-Terminal Signal Sequence Binds receptor in the surface Cleaved in the matrix or stroma Outer and inner membranes in close contact Requires energy a.a hydrophobic, positive basic a.a., hydroxylated (Ser, thr), no negatively charged a.a. Amphipathic TOM: Translocon Outer Membrane Receptor plus channel Antibodies inhibit translocation TIM: Translocon Inner Membrane Contact sites Molecular chaperon and chaperonin required for folding

To be transported the protein need to be unfolded Translocation requires ATP to maintain the protein unfolded in the cytosol and the matrix Energy as proton motive force (Electrochemical gradient)- move positive charged a.a. in transit peptide (Electrophoresed)

Transferred general import pore of the outer membrane (Tom20/22)

Path A:Stop transfer sequence block transfer through Tim23/17 channel and is moved laterally Path B: Uses Oxa1 to embed the protein into the inner membrane Path C: multipass protein with six membrane-spanning domains. No N-transit peptide Recognized by Tom70

First interaction with the general import pore (Tom40) Then Transfer to the sorting and assemble machinery complex (SAM)

Folded proteins are transported PTS in the C-terminal No removal of PTS

Nuclear pore complex Nuclear localization signal Cytosolic protein required for transport

Nuclear Import

Ras dependent nuclear export

Ras independent nuclear export