Molecular Cell Biology Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott • Lawrence Zipursky • James Darnell Molecular Cell Biology Fifth Edition Chapter 16: Moving Proteins into Membranes and Organelles Copyright © 2004 by W. H. Freeman & Company

Protein sorting in different organelles are governed by the same basic mechanisms with variations

Questions for protein sorting Sequence context of the signal sequence Identity of the receptor for the signal sequence Translocation channel structure and how protein pass it Energy source for protein translocation

Signal sequence or Uptake-targeting sequence Carrying the information for a protein to target to a particular organelle destination. Signal sequences are often removed from the mature protein by specific proteases.

Receptor proteins Each organelle carries a set of receptor proteins that bind only to specific kinds of signal sequences.

Translocation channel Translocation channel will allow the protein to pass through the membrane once a protein containing a signal sequence has been recognized by its receptor protein.

Energy Protein translocation will only go unidirectionally because it is energy-consuming.

Topics for this chapter ER protein targeting Bacterial protein export Targeting of proteins to mitochondria, chloroplast, and peroxisomes

Translocation of secretory proteins across the ER membrane In this book, secretory proteins are proteins that will be secreted or will become luminal proteins in the ER, Golgi, and lysosomes. Cotranslational translocation Post-translational translocation

Co-translational translocation of secretory proteins across the ER membrane Signal sequence: 16~30 residues with 6-12 hydrophobic residues (the core) and one or more positively charged amino acids adjacent to them. It will direct the ribosome to the ER membrane and initiates translocation of the growing peptide across the ER membrane.

Co-translational translocation of secretory proteins across the ER membrane Receptor: SRP (signal-recognition particle) and SRP receptor SRP is consisted of six proteins and a 300 nt RNA SRP receptor is consisted of two subunits, a and b (a is larger)

SRP (signal-recognition particle) It use its hydrophobic binding groove to interact with ER signal sequence.

Co-translational translocation of secretory proteins across the ER membrane Translocation channel: Sec61 complex Sec61 is consisted of three subunit, a, b and g. a is the largest, containing 10 membrane-spanning a helices. Energy source: GTP hydrolysis

Co-translational translocation of secretory proteins across the ER membrane Synthesis starts on free ribosome first. The whole complex must be translocated to ER before the 70th residue was added. Without ER, the synthesis will come to a halt.

Co-translational translocation of secretory proteins across the ER membrane GTP hydrolysis is required to increase the fidelity of this process. If the protein does not have correct signal sequence, GTP hydrolysis will occur and protein will leave the complex.

Co-translational translocation of secretory proteins across the ER membrane If the protein is correct, GTP hydrolysis will trigger translocon (Sec61) to open and protein will enter ER lumen.

Sec61 complex 5-6 nm (h) x 8.5 nm (diameter) The pore is 2 nm in diameter

Co-translational translocation of secretory proteins across the ER membrane By recognizing its C-terminal sequence, signal peptidase will cleave the ER signal sequence after it enters the ER lumen.

Co-translational translocation of secretory proteins across the ER membrane Signal sequence: 16-30 residues containing 6-12 residues of hydrophobic core with one or more positively charged residues adjacent to them Receptor: SRP and SRP receptor Translocon: Sec61 complex Energy requirement: GTP hydrolysis

Post-translational translocation of secretory protein across ER membrane Some yeast proteins are translocated into ER by this pathway. Signal sequence context is the same as co-translational translocation. Receptor: Sec63 complex and Bip Translocon: Sec61 complex Energy requirement: ATP hydrolysis

Post-translational translocation of secretory protein across ER membrane Translocating polypeptide chain will only slide back and forth without Bip, a member of Hsp70 family.

Post-translational translocation of secretory protein across ER membrane Bip hydrolyzes ATP and Bip-ADP binds translocating polypeptide so it can enter ER.

Post-translational translocation of secretory protein across ER membrane Bip-ADP pull translocating polypeptide chain into ER.

Insertion of proteins into ER membrane Main questions: How integral proteins can interact with membranes? How topogenic sequences direct the insertion and orientation of various classes of integral proteins into the membrane? Integral membrane proteins GPI-anchored proteins

Topogenic sequences Membrane-spanning segments determine a protein’s topology. 20-25 hydrophobic amino acids Form a-helix

Type I integral membrane proteins Single pass with cleaved N-terminal signal sequence C-terminal region on the cytosolic side, N-terminal region on the ER/Golgi lumen or cell exterior

Type I integral membrane proteins Sequence context is the same as the signal sequence for ER targeting Has stop-transfer anchor sequence Receptor: SRP, SRP receptor

Hydropathy profile Hydropathy profile can be used to search for topogenic sequences.

Type II and III integral membrane proteins Single pass with no cleavable signal sequence The orientations are opposite

How type II and III membrane proteins anchor differently? Positively charged residues located on one side of the hydrophobic sequences tend to stay on the cytosolic side that determines the orientation of protein.

Type IV integral membrane proteins Multi-pass Can be further categorized into IV-A and IV-B, depending on the orientation

Positively-charged segment determines the orientation of integral membrane proteins

Glycosylphosphatidylinositol (GPI)

GPI-anchored protein Some cell-surface proteins are anchored to the phospholipid bilayer by a covalently attached amphipathic molecule, GPI. Proteins with GPI anchors can diffuse in the plane of the phospholipid bilayer membrane. On the contrary, many proteins anchored by membrane-spanning a helices are immobilized in the membrane.

Synthesis of GPI-anchored proteins They are synthesized and initially anchored to the ER membrane exactly like type I transmembrane proteins. A short sequence adjacent to the membrane-spanning domain is recognized by a transamidase.

Synthesis of GPI-anchored proteins Transamidase cleaves off the original stop-transfer anchor sequence and transfers the remainder of the protein to a preformed GPI anchor in the membrane.

Export of bacterial proteins Bacterial proteins will be exported to two destinations: Inner membrane and periplasmic space Outer membrane

Export of bacterial proteins to the inner membrane and periplasmic space Signal sequence: N-terminal hydrophobic sequence (will be cleaved by signal peptidease later) Receptor: Ffh (similar to SRP) and FtsY (similar to SRP receptor) Translocon: SecY, SecE and SecG; similar to Sec61 complex Energy requirement: ATP hydrolysis

Inner membrane and periplasmic space transport is post-translational only SecA (similar to Bip) push the protein into translocon.

Translocation across inner and outer membrane to extracellular space Four types Type I and II: two-step translocation Substrate will be folded and acquire disulfide bonds in the periplasmic space first Periplasmic proteins (spanning inner and outer membranes) translocate folded proteins outside Energy requirement: ATP hydrolysis

Translocation across inner and outer membrane to extracellular space Four types Type III and IV: single-step translocation Large protein complexes spanning both membranes Pathogenic bacteria use type III for protein secretion and injection Agrobacterium tumefaciens (膿桿菌) use type IV to transport T-DNA into plant cells.

Protein sorting to mitochondria and chloroplast Mitochondrial protein targeting Mitochondrial matrix proteins Inner-membrane proteins Intermembrane space proteins Chloroplast protein targeting Stromal protein targeting Thylakoid protein targeting

Targeting for mitochondrial matrix Matrix-targeting sequence: 20-50 residues, amphipathic helix with Arg and Lys residues on one side and hydrophobic residues on the other Receptor: import receptor Translocon: general import pore (Tom40, Tim23/17 and Tim44, passive channel) Energy requirement: ATP hydrolysis

Hsc60

Mitochondrial inner membrane protein targeting Energy comes from proton motive force Three pathways: Path A use the same machinery used for targeting of matrix proteins. Tom20/22 recognize their matrix targeting sequence

Mitochondrial inner membrane protein targeting Precursors of path B contain both a matrix targeting sequence and internal hydrophobic domains recognized by an inner-membrane protein named Oxa1.

Mitochondrial inner membrane protein targeting Path C is used by multi-pass proteins containing multiple internal mitochondrial targeting sequences recognized by Tom70.

Intermembrane-space proteins targeting

Intermembrane-space protein targeting Path A is similar to pathway A for delivery to the inner membrane, but with intermembrane space-targeting sequence that will be recognized by an inner-membrane protease.

Intermembrane-space protein targeting Path B delivers protein to intermembrane space directly by Tom40.

Outer-membrane protein targeting Sequence context: N-terminal short matrix-targeting sequence followed by a long stretch of hydrophobic amino acids. Sequence will not be cleaved after translocation.

Chloroplast stromal protein targeting Sequence: no common motifs, generally rich in Ser, Thr, and small hydrophobic residues and poor in Glu and Asp. Import process is similar to mitochondria matrix protein import but import proteins are different. Chloroplast stroma does not have proton motive force, so the energy of stromal import comes solely from ATP hydrolysis.

Thylakoid protein targeting Four pathways SRP-dependent pathway SecA-related pathway Oxa1 related protein pathway ΔPH pathway

DPH pathway: unfolded proteins were transported to the stroma first, then a set of thylakoid-membrane proteins will transport them to thylakoid lumen. Thylakoid-targeting sequence contain two closely spaced Arg. SRP-dependent pathway: transport of plastocyanin (involved in photophosphorylation) to thylakoid lumen

Oxal related protein pathway Some proteins are inserted into the thylakoid membrane by this pathway.

SecA-related protein pathway This pathway transport proteins into thylakoid lumen.

Peroxisome protein targeting Sequence: PTS1 (SKL or related sequence) near the C-terminus is required for peroxisome targeting, it will not be cleaved after transport ATP hydrolysis is required. Receptor: Pex5, Pex14 Translocon: Pex10, Pex12 Folded proteins can be translocated.

Principle modification for proteins Glycosylation: ER, Golgi Disulfide bond formation: ER Polypeptide chain folding and multisubunit assembly: ER Proteolytic cleavage: ER, Golgi, secretory vesicles

Glycosylation Glycosylation help folding, promote cell-cell adhesion and confer stability. O-linked glycosylation can be found in collagen and glycophorin, containing only one to four sugar residues. N-linked glycosylation is more complex but more common.

N-linked glycosylation The 14-residue oligosaccharide precursor for N-linked glycosylation is synthesized in ER and will be modified in ER and Golgi later.

The oligosaccharide precursor The three glucose (glc) residues serves as a signal that this precursor is complete and is ready for transfer.

N-linked glycosylation The 14-residue precursor will be transferred from dolichol to the asparagine residue of the target protein with Asn-X-Ser or Asn-X-Thr sequence by oligosaccharyl transferase. Then three glc and one man will be removed.

Disulfide bond formation In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER; in bacterial cells, disulfide bonds are formed in the periplasmic space between the inner and outer membranes. Thus, disulfide bonds are found only in secretory proteins and in the exoplasmic domains of membrane proteins.

Disulfide bond formation Protein disulfide isomerase (PDI) can catalyzed the formation of disulfide bond and can correct wrongly-formed disulfide bond.

PDI will be oxidized by Ero1 How Ero1 become oxidized again is not clear….XD

Polypeptide chain folding Both BiP and PDI can help protein folding. Calnexin and calreticulin will bind selectively to certain N-linked oligosaccharides on growing nascent chains, preventing aggregation of adjacent segments of a protein as it is being made. Peptidyl-prolyl isomerases will accelerate protein folding by rotating peptidyl-prolyl bonds.

BiP and PDI help protein folding

Peptidyl-prolyl isomerase The rotation of peptidyl-prolyl bonds sometimes are the rate-limiting step in the folding of protein domains.

The unfolded protein response