Molecular Cell Biology Fifth Edition
Chapter 16: Moving Proteins into Membranes and Organelles So far, two fundamentally different ways of translocating proteins are known: Chap. 16: proteins are delivered in soluble forms. Chap. 17: proteins are delivered in vesicles.
Harvey Lodish • Arnold Berk • Paul Matsudaira • Chris A. Kaiser • Monty Krieger • Matthew P. Scott • Lawrence Zipursky • James Darnell
Hsou-min Li
2006 1
Why do we have the problem of “protein sorting”? -- proteins are made at the same place but have to be sent to different organelles.
only one kind of cytosolic ribosome
Generally divided into: 1. secretory pathway :cotranslational 2. organelle biogenesis: post-translational Common mechanism for protein sorting to all organelles: 1. A targeting sequence 2. A receptor (complex) Organelle biogenesis 3. A translocation channel across the membrane 4. An energy favorable system to drive the transport and make the transport unidirectional So for all the systems we talk about today, we will ask 4 questions: 1. What is the nature of the targeting signal? 2. What is the receptor? 3. What is the structure of the channel? 4. What is the source of energy?
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16.1 Translocation of secretory protein across the ER membrane
We will discuss: 1. signal peptide 2. SRP 3. SR and the GTP cycle 4. Sec61p channel 5. Post-translational translocation 6. Membrane protein topology
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But let’s first talk about some old history: How did we know secretory proteins don’t just directly cross the plasma membrane from the cytosol? In addition: Import into ER has to be co-translational. Because: Secretory proteins are localized in the ER lumen shortly after synthesis
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How was the pathway defined? a. Gorge Palade: pulse-chase with pancreatic cells (by injecting guinea pigs with [3H]leucine) 1974 Nobel Prize of Physiology and Medicine (b. genetic evidence and c. glycan processing in Chapter 17)
3 min after chase: ER
37 min: immature secretory granules
7 min: Golgi
117 min: mature secretory granules 5
1. ER signal peptide
Mutations that disrupt the hydrophobic core disrupt translocation Many artificial sequences containing a hydrophobic core can function as an ER signal sequence6
2. SRP (signal recognition particle) identification: high-salt washed ER membranes no longer function in transport --add back the wash solution and the activity was restored. One RNA, 6 proteins. Different domains have different functions 3. SR (SRP receptor) and the GTP cycle SR identified by protease treatment of ER membrane. Reconstitution: only need ribosome/nascent chain complex, SRP, SR and the Sec61p complex and GTP. Therefore the only energy required is GTP, for SR/SRP and for translation (“pushing”).
membrane bound
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4. Sec61p channel Identified by yeast mutants (that fail to secret) and confirmed by cross-linking experiments. Composed of three proteins Sec61 !, ", #, with Sec61! being the major component.
How does the channel open and close? Still controversial, at least three hypotheses 1. Always open, sealed by Bip and ribosome 2. Pore formed by assembling 4 complexes upon translocation 3. Diaphragm
1. Always open, sealed by Bip and ribosome
JCB vol 156, p218, 2002
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2. Pore formed by assembling 4 complexes upon translocation The 1975 signal hypothesis predicted: 1. an N-terminal localized targeting signal on the protein itself. 2. a binding factor that guides the protein to ER (deleted in the formal hypothesis, sigh!) 3. a signal-induced protein-conducting tunnel through the ER membrane.
1971
Biomembranes 2 p193-195 (1971)
1975 The picture shows pores formed by 4 trimers (!, ", #). Cell Vol 87 number 4, 1996
JCB 67, p835-851 (1975)
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3. Diaphragm 2004 channel crystal structure: a plug formed by one of the ! helixes in the sec61! subunit. The plug moves away when signal peptide binds.
!"#$%&'"()*(+,-$./*%(+-0"#++%1 !"#$%&'()*+,&*)$&!",)&-.&/0)1
2%314-5#6%-$./*%(+'-#.%-5/7%6-#0./''-0%1181#. 5%59.#+%'-*"./8,"-#-$./*%(+-0"#++%1:-;"%-0.4'*#1 '*.80*8.%-/)-*"('-0"#++%1-('-+/3-.%7%#1%6-#+6 0/+)(.5'-%<$%0*#*(/+'-*"#*-(*-58'*-0"#+,%-'"#$%-*/ #11/3-$./*%(+'-*/-$#'': The isoleucine ring in the center of the hour-glass shaped channel is too small for polypeptide chain to pass. It is predicted that the backbones of the channel have to move like diaphragm to increase the pore size. 10 2=;>?@-ABC-DEF--G-H=2>=?I-EJJD-$ED&EKL-$MK&DD
Nature vol 427 p 36-44 2004
The minimum unit is one copy of the heterotrimer. 10 !-helix trans-membrane domains of Sec61! form the main body of the channel. One of the alpha helixes of the alpha subunit forms the plug. There is a hydrophobic pore ring in the center of the channel. Membrane proteins can diffuse laterally into the lipid bilayer. The pore seen in the previous EM was probably space among 11 several actual channels.
5. Post-translational translocation: Some proteins are fully translated before being translocated across the ER membrane. Fairly common in yeast, occur occasionally in higher eukaryotes. Do NOT need: SRP , SR nor GTP. Need: Sec61 complex, Sec63 complex, Bip and ATP
Polypeptide slides inward and outward (Brownian motion).
“Brownian ratchet” Interaction of Bip-ATP with Sec63 (containing a J domain) causes ATP hydrolysis by Bip.
Bip binding prevents backward sliding of the polypeptide.
Bip-ADP binds stably to unfolded polypeptides.
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16.2 Insertion of proteins into the ER membrane Topology: number of times a membrane protein spans the membrane and the orientations of the membrane spanning segments Topology of all membrane proteins in the secretory pathway is determined during insertion into ER. Importance: proper function depends on correct topology Q: which side will be intracellular, which side will be extracellular?
Topology is mainly determined by the direction of insertion of the first transmembrane domain (usually the signal peptide), and whether the signal is cleaved. The rest of the transmembrane domains insert accordingly. You can flip the entire orientation by changing the charges flanking the first transmembrane domain. The “positive inside” rule 13
N N
+
+N
+
N
+ N
N
N
N
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Topology prediction from sequence 1. To predict number of transmembrane domains: Hydropathy profile (hydropathy plot) first assign a value of hydropathic index to each amino acid then calculate the sum of 20 (of some other number) consecutive amino acids along the entire length of the polypeptide. To predict orientation: positive inside rule 2. Sequence homology to other knows proteins
GPI (glycosylphosphatidylinositol) anchored proteins This end won’t be interacting with other cytosolic proteins or cytoskeleton, so can diffuse more freely. Why are all GPI-anchored proteins extracellular surface proteins?
Synthesized as Type I ER membrane protein
GPI transamidase cleaves the membrane anchor and transfer the soluble domain to preformed GPI
Another function: apical sorting: GPI anchor targets the attached protein to the apical domainz of the plasma membrane in certain polarized epithelial cells. 15
16.3 Protein modifications, folding, and quality control in the ER Types of modification: Glycosylation (addition and trimming) proteolytic cleavage (processing)
In both ER and Golgi
S-S formation Only in ER Folding Multi-subunit assembly I. Glycosylation: mostly in ER and Golgi. Most Golgi resident proteins are glycosyltransferases. Very few cytosolic proteins are glycosylated (a few transcription factors are). Recently identified: a transport pathway from cis Golgi to chloroplasts and some chloroplasts proteins are glycosylated. By vesicles? Or soluble proteins? Or something totally different? Two types, N-inked and O-linked
O-linked: to -OH groups of serine and threonine. One-by-one added from sugar nucleotides in ER, cis-Golgi and transGolgi.
trans-Golgi. ER, cis-Golgi
N-linked: to amide nitrogen of asparagine in the sequence Asn-x-Thr or Asn-x-Ser. Synthesized also from sugar nucleotides, added in ER first as a pre-assembled big block of 14 sugars then diversely modified in ER and Golgi. sugar nucleotide precursors
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Functions of the glycan groups 1. Promote proper folding (through calnexin, see next page): mutation or treatment with tunicamycin to inhibit glycosylation can cause misfolding of some proteins 2. Promote stability in blood of many secreted glycoproteins 3. Cell-cell adhesion: e.g. glycan on CAM (cell adhesion molecules) of leukocytes binds to sugar binding domains of CAM of endothelial cells lining blood vessels. 4. Major antigens: ABO blood types (O-linked glycan attached to glycoproteins and glycolipids on the surface of erythrocytes.
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II. Disulfide bond formation: by Ero1, protein disulfide isomerase (PDI) and unknown oxidant. Only in ER lumen and bacterial periplasmic space. So only secreted proteins or extracelluar domain of membrane proteins have S-S. E. coli over-expressed proteins often aggregate due to lack of S-S formation during synthesis
S-S is often re-arranged in ER lumen by PDI
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III: proper folding and multimeric protein assembly (in ER): Proteins that assist folding in ER Hsp70 class chaperone: e.g. Bip PDI!S-S calnexin and calreticulin: bind to glycans thus prevent folding of adjacent amino acids so PDI and glycosyltransferases and other chaperones have time to properly fold the polypeptide. Peptidyl isomerase: accelerate the rotation about a peptide bond (cis <--> trans) A good example that contains all the modifications: hemaglutinin (the spikes protruding from the surface of an Interaction of the membrane spanning !-helix from three influenza virus) assembly subunits triggers the formation of a long stem containing one ! -helix from the luminal part of each subunit.
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In the Golgi, more glycan processing
Some proteins undergo proteolytic processing after leaving the TGN: e.g. the processing to proinsulin to insulin takes place in the secretory granules.
Why don’t secretory proteins just directly cross the plasma membrane from the cytosol?-- they have to go through the processing/modification factory of ER and Golgi to make sure they are properly modified and folded.
What if they can’t be properly folded ?? 20
IV. The unfolded-protein response (Figure 16-22): Accumulation of unfolded or misfolded protein in the ER lumen triggers the increase in transcription of genes encoding ER chaperones and other folding catalyses.
This splicing occur in the cytosol HAC1 is transported back to the nucleus and activates the transcription of ER-localized chaperones to participate in binding of unfolded proteins.
Only properly folded and assembled proteins get to leave ER.
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V. quality control: Most mis-folded proteins are transported back out the ER (some through the Sec61p translocon, some through a newly identified channel called Derlin-1) and degraded by the ubiquitin proteosome system in the cytosol.
a virus protein (to destroy cell’s antiviral defenses)
Figure 6. Model for US11-mediated retrotranslocation of MHC class I heavy chains. US11 recognizes HC in the ER lumen and targets it to Derlin-1, a proposed component of the retro-translocation channel. The p97 ATPase complex is recruited to Derlin-1 by VIMP. HC emerging into the cytosol is bound by p97. Poly-ubiquitin chains (PolyUb, red) are attached and recognized by both the N-domain (N) of p97 and the cofactor Ufd1/Npl4 (U/ N). ATP hydrolysis by p97 moves HC into the cytosol. The retro-translocation of misfolded ER proteins may occur similarly, with US11 being replaced by other targeting components. NATURE |VOL 429 | 24 JUNE 2004 p841-847
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16.4 Export of bacterial proteins--all post-translational Two major parts: 1. Across the inner membrane: to be inserted into the inner or outer membrane or trapped in the periplasmic space. 2. Across both the outer and inner membranes and to be secreted the extracellular space or injected into other cells 1. Across the inner membrane: similar to ER membrane translocation (but post-translational) So for all the systems we talk about today, we will ask 4 questions: 1. What is the nature of the targeting signal? Similar to
ER signal peptide (inter-changeable), cleavage by signal peptidase (also inter-changeable). Homologues for SRP and SR are also involved, but only used for post-translational insertion of very hydrophobic membrane proteins into the inner membrane. 2. What is the receptor? SecA (and SecB) 3. What is the structure of the channel? SecYEG,
similar to Sec61!"# Current Sec61 structure is actually from an archea. 4. What is the source of energy? ATP,
not GTP
cytosol periplasmic space
S-S bond formation and folding
SecA uses the energy of ATP hydrolysis, which causes a conformational change in SecA, to actively push the polypeptide through the SecYEG channel. A pulling mechanism from the periplasmic space won’t work because ATP will diffuse away through the outer membrane.
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2. Across both the outer and inner membranes and to be secreted into the extracellular space or injected into other cells: divided into 4 types (I, II, III, IV) Type III: Used by some pathogenic bacteria to inject toxins into host cells. 1. What is the nature of the targeting signal? An
amphipathic sequence at the N terminus (for YopE) 2. What is the receptor? Small chaperone proteins 3. What is the structure of the channel?A syringelike structure with similarity to bacterial flagellum 4. What is the source of energy? ATP Needle complex structure by cryoEM at 17 angstron from Salmonella typhimurium Fig. 1. The needle complex and the base complex of the TTSS from S. typhimurium can adopt different symmetries in vivo. (A) Nomenclature of the structural features of the needle complex.The needle complex is divided into two distinctive substructures: the membrane-embedded base and the extracellular needle filament. The base spans the periplasm and is associated with the inner and outer membranes, where ringlike structures are visible in electron micrographs of negatively stained needle complexes (2% phosphotungstic acid, pH 7) (B). The outer membrane– associated rings (OR1 and OR2) are composed of the protein InvG, and the inner membrane– associated rings (IR1 and IR2) contain the proteins PrgH and PrgK (4). The only protein identified for the needle filament to date is PrgI (4). Bar, 30 nm. (C) Model-based multireference alignment revealed significant differences in the diameters of the average projections obtained for different rotational symmetries, as indicated by white arrows in the comparison of the IR1 of the 19- and 22-fold particles. (D) Distribution of different symmetries in needle complexes isolated from wildtype S. typhimurium. The data were generated by examining 3577 particles. (E) After sorting of the particles and 3D reconstruction without enforcing any symmetry, the true rotational symmetries could be derived from cross sections through IR1 of the reconstructed needle complexes, as shown for the 20- and 21-fold particles.
5 NOVEMBER 2004 VOL 306 SCIENCE p1040-1042
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16.5 Protein sorting to mitochondria and chloroplasts--all post-translational.
Both organelles have N terminal cleavable signals. Comparing with the other two signals:
Mitochondrial targeting signal: amphipathic !-helix (positively charged amino acids on one side, hydrophobic amino acids on the other side. For “electrophoresis”+ across the inner + membrane +
o Hydrophobic o interaction with o Tom20
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Receptors/ channels: Tom/Tim (translocon of the outer/inner membrane) First identified by antibody inhibition, then by genetic (confirm previous ones with knockout, identify new ones by suppressor), now by mass spec. or genomic approach. General outline of the import process e.g. for matrix proteins: Tom20/Tom22 receptor--> Tom40 channel-->contact site-->Tim23/Tim17 channel-->Hsp70/Tim44 ratchet Several important features: 1. Precursor translocated in unfolded form 2. Translocate through contact site 3. Energy: ATP on the outside for unfolding, in the matrix for ratcheting precursors. PMF (400,000 V/cm) at the inner membrane for “electrophoresis” across the inner membrane
Tim50
+++
__
_
Translocate through contact site
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Current model
Evolution of the Molecular Machines for Protein Import into Mitochondria Pavel Dolezal, Vladimir Likic, Jan Tachezy, Trevor Lithgow
SCIENCE (2006) VOL 313 p314
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Multiple signals and pathways targeting proteins to submitochondrial compartments
Tom70
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Protein import into chloroplasts Similarity to mitochondria a Toc (translocon of outer membrane of chloroplasts) complex with a receptor and a channel a Tic complex with a connector and a channel Energy: ATP in the intermembrane space and stroma, possibly for Hsp70 but not sure yet. GTP is only demonstrated by GTP-r-S inhibition. 4 pathways to thylakoid, 3 have bacterial homologous pathways 2 for thylakoid lumen: $pH (Tat) for folded protein, use $pH SecA for unfolded protein, use high ATP and SecY 2 for thylakoid membrane SRP: for LHCP, use GTP as energy and SecY and Alb3(Oxa1) as membrane receptor spontaneous
Biochem. Cell Biol. 79: 629–635 (2001)
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | MAY 2001 | 350-356
SRP/SR for posttranslational insertion of very hydrophobic membrane proteins into the inner membrane.
29 Traffic 2001; 2: 245-251
16.6 Protein import into peroxisomes: single membrane, no DNA, catalase as marker enzyme, most abundant in liver (1-2 % of the liver cell volume). 1. What is the nature of the targeting signal? For most matrix proteins: PTS1: C-
terminal SKL, not cleaved after import. For a few other matrix proteins: PTS2, approx. N terminal 9 amino acids, may or may not be cleaved. Membrane proteins use different system (see below). 2. What is the receptor? Pex5 (PTS1), Pex7(PTS2) converge on the membrane docking site Pex14/13/17, structure unclear. Pex5 partially or fully enters the matrix, releases the cargo then recycle back to the cytosol. 3. What is the structure of the channel? Pex 10/12/2, structure not clear. Peroxisome can import folded proteins,
even 4–9 nm gold beads coated with albumin bearing PTS1s and microinjected into cultured cells. However, no pore structure like the nuclear pore exists. Require transporter proteins for ions and proton passages--so it’s totally sealed. 4. What is the source of energy? ATP, but don’t know for what.
?
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2 pathways to produce new peroxisome: de novo synthesis, and division (other organelles like mitochondria and chloroplasts do not have de novo synthesis)
de novo (Probably from ER membranes)
division
Peroxisome membrane and matrix proteins are imported by different pathways
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