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Subcellular compartments and protein targeting Alex van der Bliek. Cellular compartments. TGN. early endosome. Golgi. peroxisomes. late endosome or MVB. ERGIC. ER. lysosome. mitochondria. Compartments allow for: Specialized environments Vectorial processing
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Alex van der Bliek
Compartments allow for:
Proteins are synthesized on polysomes
RM = rough
No SRP, no arrest
Residual SRP allows some translocation
Too late for SRP
K-RM = rough microsomes
stripped by washing with
KCl, which removes SRP
Delay in adding RM
causes a buildup of arrested product.
Once RMs are added the arrested products are converted to mature prolactin.
70 aa leader = arrested product, consisting of signal peptide + 40 aa buried inside the ribosome
Classic paper by Walter and Blobel (1981) J. Cell Biol. 91, 557-561.
SRP receptor = heterodimer with two GTPases (SRa and SRb)
SRa and SRP54 bind to each and act as others GAPs. GTP hydrolysis provides direction
Then the ribosome is passed on to the Translocase
Identified by Schekman with yeast genetic screen:
Result of the screen: mutants called sec61, sec62, sec63, etc.
Sec61 is the translocase: a 10 pass membrane spanning protein that forms a trimer.
sec62 sec63 sec61 TRAM
Method of protein insertion
Cytoplasm C N N
ER lumen N C C
type I type II multipass
Signal peptidase cleaves signal peptide in ER lumen
Nascent protein is sequestered by chaperones
Hsp70 - first line of defense against aggregation, Hsp70 in the ER lumen is called BiP (binding protein)in mammals or Kar2 in yeast. BiP is essential for translocation
Protein disulfide isomerase (PDI), Erp57, etc.
ER lumen is oxidative -> disulfide bonds form spontaneously. Misfolded proteins need to be rearranged by PDI and ERp57
Prolyl peptide isomerase (PPI)
Cis-trans isomerization of prolines is normally very slow
Calnexin and calreticulin
Calcium binding proteins in ER that function as chaperone., Bind and retain incompletely glycosylated proteins.
TAP (Transport of associated with antigen processing)
Antigenic peptides made by the proteasome
Normal processing pathway of MHC Class I
mature Class I
ERAD: ER associated degradation
It was long thought that sec61 is involved, but in 2004 Derlin-1 and Derlin-related proteins were implicated. Is sec61 still involved?
What are the minimal requirements for retrotranslocation?
Fig. 1. An in vitro assay for retrotranslocation.
Microsomes were preincubated with radiolabeled (35S-met) fluorescent (BOF) and glycosylation defective prepro-a-factor (pDgpaf-BOF).
BOF is a fluorescent Lys-tRNA added through in vitro translation.
Retrotranslocation is detected by quenching with a fluorescence quencher added to the cytosol.
First, pDgpaf-BOF is imported into microsomes and the signal peptide is removed upon import (Dgpaf-BOF)
Then, retrotranslocation is started by raising the temperature at 0’.
Retrotranslocation requires cytosol.
Overall decrease in radiolabel is though proteasome (Lactacystin shown in Suppl. Fig.)
Panel B: Fluorescence
Fig. 2. Release of Dgpaf-BOF from microsomes with defined lumenal contents
Panel A: Microsomes were extracted using high pH and resealed in the presence of Dgpaf-BOF and other defined components
Panel B shows a bump, followed by gradual decrease in -cyto and -ATP, also seen with control (non-translocated) substrates, such as PDI and BiP.
Panel C is corrected for this decrease.
l = anti-BOF antibody, which quenches the fluorescence
cpt = complete): resealed with original lumenal content.
RRMs = reconstituted rough microsomes.
cyto = cytosol from which haemoglobin was removed
-ATP = preincubated with apyrase
epoxomicin = proteasome inhibitor
F = F+aBOF/F-aBOF F0 = F at 0’
Panel D: SDS gel recovers fluorescence from supernatant = direct biochemical evidence for release
Conclusion: It is possible to reconstitute the reaction.Release requires cytosol and ATP.
p97 (Cdc48), Npl4 and Ufd1 (proteins that transfer poly-ubiquitinated proteins to the proteasome) are ineffective.
NEM is an alkylating agent used to inactivate the 19S particle (regulatory subunit of the proteasome)
As in panel C shown on an expanded scale
26S (the complete proteasome) does work
Conclusions: the proteasome is the only cytosolic component needed for retrotranslocation
Panel A: some translocation occurs without lumenal content, Bip helps, but PDI is the best, just as good as complete
Panel B: PDI increases Dgpaf-BOF anisotropy, suggesting that they bind, even though Dgpaf-BOF lacks cysteines
Panel C: oxidized PDI works better, even though it binds more poorly to Dgpaf-BOF
Wikipedia: Fluorescence anisotropy assays rotational diffusion using decorrelation of polarization in fluorescence (exciting versus emitted photons). This helps estimate the shape/size of the molecule.
Conclusions: PDI is needed to fully reconstitute retrotranlocation
Background: Retrotranslocation was originally thought to involve sec61, but in 2004 Derlin-1 and related proteins were shown to be involved. Does sec61 still contribute to this process?
a-sec61 antibodies have no effect
a-Der1 antibodies do inhibit release
Expanded panel C showing that blockage occurs after a 2 min lag
RNC (ribosome with nascent chain) and mutant BiP also have no effect
Conclusions: Der1, but not sec61, is required for retrotranslocation.
Dgpaf was synthesized with a photoactivatable cross linker (ANB) and incoporated in RRMs.
The reaction went for 0 or 15’ before UV cross linking.
Samples were solubilized with detergent and coIPs were conducted with Der1, sec61 and TRAM antibodies.
The image shows an autoradiogram.
1. unidientified species
2. Dgpaf crosslinked to Der-1
3. Dgpaf alone
Conclusions: Dgpaf can be cross-linked with Der1, but not with sec61 or TRAM
On the lumenal side, PDI is enough for retrotranslocation.
Derlin is required for retrotranslocation, but sec61 is not.
On the cytosolic side, the proteasome is enough for retrotranslocation.
Retrotranslocation complex contains derlin (reviewed in Cell 126, 237-9, 2006) but other proteins may also contribute in vivo.
Import into Mitochondria and peroxisomes
Pellet (mitochondria) = protease insensitive
Mitochondria (Double membrane - Related to bacteria - Endosymbiont)
Mitochondria have their own DNA, transcription and translation machineries, but encode very few proteins (13 in mammals).
Most mitochondrial proteins (1,500) are encoded by nuclear DNA.
Synthesis is on free polysomes and import is post-translational
Mitochondrial targeting signals:
N-terminal amphipathic helix
or internal sequences.
Nascent mitochondrial leader sequences bind to MSF (mitochondrial stimulation factor), a chaperone that passes them on to the import machinery.
In vitro assay for mitochondrial import (Schatz and Neupert, early 80s):
+ + positively charged + +
Cytosolic Hsp70 and MSF keep the cytosolic precursors soluble.
Tom70 and Tom 20 receptors for mito-leaders on the mitochondrial surface.
Tom40: outer membrane import channel
Tim23: inner membrane import channel
Tim44, Hsp70 and GrpE drive translocation by ATP hydrolysis.
Mitochondrial Hsp70, GrpE and hsp10/hsp60 facilitate folding.
MPP (Mitochondrial Protein Peptidase) cleaves the mito-leader.
Nature 424, 565-571.
Panel a: Mas37 deletion slows progression of Tom40 from precursor, through assembly complexes I and II to mature Tom complex. Assembly complex I has an aberrant size (*).
Panel b: The amounts of Tom40 and porin (2 outer membrane proteins) are decreased, but the amounts of other proteins are not (IMS, inner membrane and matrix). (Western blots)
Method: In vitro translate Tom40 with 35S -Met and incubate with purified mitochondria
Solubilize with digitonin and analyze with Blue Native gel (BN-PAGE) and autoradiography
Follow progression of Tom40 through intermediate complexes with time course.
Fig. 1. Panel c. Import of inner membrane and matrix proteins is not impaired in a mas37 deletion.
Isolated mitochondria were incubated with radiolabeled precursors with or without inner membrane potential (DY)(required for import into matrix). The mitochondria were then treated with or without proteinase K to remove protein from the surface.
p, precursor; I, intermediate; m, mature
Fe-S, F1b, a-MPP b2D-DHFR and Su9-DHFR are all proteins imported into the matrix.
AAC and PiC are inner membrane proteins.
BN-PAGE shows that AAC and PiC still dimerize in a mas37 deletion.
The amount of Tom complex decreases when the mas37D strains is grown at elevated temperatures.
The amount of Tom40 compared with the amount of mtHsp70 (a matrix protein).
Overall conclusion from Fig. 1: Mas37D affects Tom40 import, but not or to a lesser extent import of matrix and inner membrane proteins. Imprt is halted priuot to the formation of assembly complex I.
Panel a. Antibodies against Mas37 inhibit Tom40 import, but not the import of AAC or F1b
AAC: BN-PAGE, autorad
Tom40: BN-PAGE, autorad
F1b: SDS-PAGE, autorad
F1b: SDS-PAGE, quantitation
Panel b. Cross-linking with EGS shows a transient interaction between Tom40 and Mas37.
Mitochondria were incubated with 35S-Tom40, cross-linked at 5 or 60 min, solubilized and subjected to immuno-precipitation with mas37 antibody or preimmune serum.
The red arrow points to the product of crosslinking, which exists at 5 min, but not at 60 min.
Panel c. Porin assembly is also affected in mas37D mitochondria.
(Porin trimerizes and then associates with several other proteins.)
SDS-PAGE, autorad, protease
Panel d. The Mdm10 complex is also affected by Mas37D.
Mdm10 is a mitochondrial outer membrane protein in a complex with Mdm12 and Mmm1. These affect mitochondrial morphology, but subsequent papers show that they are also part of the new import pathway uncovered with Mas37.
Panel e. The Ugo1 complex is not affected by Mas37D.
Ugo1 is in another mitochondrial outer membrane protein in a complex with Fzo1. They affect mitochondrial fusion. There does not seem to be a connection with the new import pathway uncovered with Mas37.
Incubate mitochondria with35S-Mdm10 or 35S-Ugo1, solubilize the protein complex with digitonin, subject to BN-PAGE, autoradiography
Panel a. Mitochondria were incubated for 5 min with 35S-Tom40 . Assembly was allowed to continue, but halted at different stages with temperature shifts.
A series of antibodies were added prior to lysis with digitonin. These antibodies cause a shift on BN-PAGE, when Tom40 is in the corresponding complex.
Assembly complex I
Assembly complex II
Panel b. SAM complex is in the mitochondrial outer membrane.
Mitochondria were isolated by differential centrifugation.
The outer membrane was ruptured by hypotonic swelling.
Outer membrane shards reseal to form vesicles (OMV).
The density of the mitochondrial outer membrane is lower than that of the inner membrane (fewer proteins). This allows for separation with a sucrose gradient.
The upper panel shows WB of SDS-PAGE probed for markers for outer membrane (Tom40), intermembrane space (cyt. C), inner membrane (AAC, Tim23) and matrix (mtHsp70).
The lower panel shows WB of BN-PAGE probed for the TOM and SAM complexes
Conclusion: SAM and Mas37 are in outer membrane vesicles (OMV).
Effects of mutations in Tom proteins on the SAM complex.
Tom22 and Tom6 deletions affect the size of the Tom complex, but Mas37 deletion does not.
Fig. 3. Continued.
The abnormal complex (SAM*) formed in a Tom7 mutant is protease sensitive
The SAM complex is protease resistant
Mas37 is part of SAM and SAM*
Mas37 is nevertheless cleaved by Prot K
Panel a: Tom mutant mitochondria are impaired in formation of assembly intermediate I, yet do not accumulate non-assembled Tom40.
(Mas37D mitochondria do accumulate non-assembled Tom40)
Conclusion: Tom complex is needed for the initial import.
Panel b: Tom40 fragment generated by trypsin is not substantially altered in Mas37D mitochondria, suggesting that Mas37D does not affect the initial import and folding of Tom40.
Panel c: Accumulation of two-membrane-spanning b2D-DHFR/methotrexate, followed by import of 35S-labeled precursors and protease treatment.
b2D-DHFR is the mitochondrial leader sequence of cytochrome b2 fused to DHFR. When incubated with methotrexate, the DHFR moiety becomes locked in the folded state, jamming both outer and inner membrane import channels.
However, the Tom complex is 4 X more abundant than Tim complex, so porin is still imported. Hence the test with tom40-6 (a mutant allele).
Panel d: Protein levels of tom40-6 mitochondria.
The amount of Tom complex is reduced in Tom40-6, but the steady state levels of various marker proteins are the same.
Panel e: Inhibition of Tom40 assembly by accumulated b2D-DHFR/methotrexate in energized tom40-6 mitochondria.
The inner membrane potential (Dy) is need for import into the inner membrane. Blocking import with b2D-DHFR/methotrexate requires Dy.
Panel f: Assembly of Tom40 in tom40-6 mitochondria is not inhibited by addition of b2D-DHFR/methotrexate in the absence of a Dy.
Mas37 is part of a new import pathway for mitochondrial outer membrane proteins, such as porin and Tom40.
This pathway uses the Tom complex for initial import, but then the newly imported proteins are passed on through two other complexes (Assembly complex I and II) before they are released as newly formed protein complexes.
After this paper was published, another component of the assembly complexes was discovered: Sam50.
Sam50 is similar to bacterial proteins that are required for insertion of b-barrel proteins in bacterial outer membranes. Sam50, porin and Tom40 are the three known b-barrel proteins in mitochondrial outer membranes.
(A paper in last months JCB showed that several other mitochondrial outer membrane proteins also use the SAM pathway).
From a review in JCB 179, 585-591, 2007
Single membrane organelles, sequester b oxidation of fatty acids and catalase.
Patients with Zellweger’s syndrome: defect in protein import (peroxisome ghosts)
Mammalian cells luciferase catalase
(Subramani lab) wt
catalase = control
punctate = peroxisomal
diffuse = cytosolic DC12
C-terminal deletions give diffuse cytosolic staining (cytoplasmic).
The import signal proved to be C-terminal SKL (PTS1)
A few peroxisomal proteins have anN-terminal cleaved leader(PTS2)
pex mutant: Subramani identified possible receptor proteins by screening for yeast mutants that fail to grow on oleic acid. These mutants delineate import pathway.
Bulkflow: ER Golgi outside
But some proteins stay in the ER (BiP, PDI, etc.): What keeps them there?
Retrieval from cis-Golgi or exclusion from transport vesicles?
ER-retention signals found by sequence comparisons:
Lumenal proteins have C-terminal: -KDEL (mammals)
-HDEL (S. cerevisiae)
Transmembrane proteins have: -KKXX or -KXKXX
Experimental evidence: transfect mammalian cells KDEL-tagged lysozyme and observe with fluorescence microscopy (wildtype lysozyme is normally secreted)
wt lysozyme lysozyme + KDEL
BiP DKDEL wt BiP
Conclusion: KDEL is necessary
and sufficient for ER retention
Yeast screen for HDEL receptor
Yeast cannot take up sucrose; Invertase is needed for growth on sucrose.
Invertase is secreted into the periplasm and converts sucrose to fructose and glucose
Add HDEL to invertase:
Invertase-HDEL is stuck in ER
Mutagenize the yeast and screen for colonies that grow on sucrose
Analyze for ER retention defect.
RESULT: ERD1 = pleitropic (no good)
ERD2 = specific ER retention defect (good)
Immunofluorescence shows ERD2 is primarily localized to cis-Golgi.
ERD2 lies in waiting to catch ER proteins that have escaped.
What sends ERD2 back to ER?
Mutations in Asp197 prevent retrieval, but not binding to KDEL.
This side of the helix may interact with other proteins to send ERD2 and the KDEL protein from Golgi to ER.
Conclusion: ERD2 provides a mechanism for retrieval of soluble ER proteins
What regulates vesicle budding?
How do vesicles bud and get severed?
How do vesicles find their target?
How do vesicles fuse?
Membrane traffic is studied with cellular, biochemical and genetic techniques. An important advance was the development of in vitro assays. These assays require:
Intact organelles. Gentle lysis can be achieved by:
- osmotic swelling and scraping
- peeling off of membranes
- slow freeze/thaw
Markers, altered by transport
- EndoH resistance
- 3H-GlcNAc incoroporation
- prepro-a-factor sedimentation
“cis-medial Golgi complementation assay”
Golgi isolated from glycosyltransferase mutant cell line
Mutant cells express VSV-G protein, but lack the enzyme that incorporates 3H-GlcNAc
Golgi isolated from wildtype cells
Medial Golgi from wild type cells can complement the glycosylation defect upon successful transport
Donor, ATP, yes
cytosol acceptor + cytosol
Donor, ATP no
NEM-cytosol acceptor + cytosol
Donor, ATP no
Rothman’s assay was used to isolate vesicular transport proteins.
NSF = NEM Sensitive Factor
NEM inactivates proteins by
Mild NEM-treatment of cytosol
inactivates a single protein
needed for transport.
NSF was the first of a series
proteins involved in transport.
GTPgS also blocks transport
Small non-clathrin coated vesicles accumulate in Golgi preparations.
Bovine brain cytosol
No vesicles (donor + acceptor)
Coated vesicles (+GTPgS)
COPs (coat proteins) form a 600 kD complex (coatamer).
The sequence of b-COP is similar to that of b-adaptin.
ARF (ADP-Ribosylation Factor), first identified as the target of cholera toxin.
part soluble, part myrisoylated
COPs abundant, soluble, in a
600 kd complex called a coatamer
ARF + coatamer + Golgi membrane is sufficient for budding
and the stoichiometry of ARF : coatamer = 3 : 1
This suggests that ARF is a structural part of the vesicle coat
ARF-GEF (trigger for budding?)
Schekman (early 80s)screened for yeast mutants with a secretion defect
This screen was based on the finding that secretion defects cause higher density
Since the Sec mutants accumulate large amounts of protein
Initial screen found mutations in 23 different genes (now over 60)
early acting (ER, Golgi)
Preliminary ordering of the mutants (accumulate core glycosylated proteins)
late acting (plasma membrane)
Schekman initially focused on early stage mutants:
accumulate excess ER
50 nm transport vesicles
Genes were ordered further with double mutants
sec17 = SNAP
sec18 = NSF
sec22 = synaptobrevin
sec21 = bCOP
sec21 ?? sec21 ??
ER vesicles Golgi
Organelles were isolated from yeast by slow freeze/thaw of spheroplasts
Marker ER transport vesicles
Post translational vesicle formation
Supernatant low speed pellet high speed pellet
With this assay Schekman started adding back cytosolic components
to determine what is required for vesicle formation from the ER
Puzzle: b-COP (sec21) is not required for budding.
But what is required?
Answer: A new coat complex, that they called COPII
COPII has 6 proteins:
GTP binding protein sar1
GEF, activates sar1 sec12
Structural component sec13
Structural component sec31/p150
GAP, inactivates sar1 sec23
Helps uncoating sec24
Flattened lattice showing sec13 and sec31
connections in a COPII coat
Structure of the COPII cage (cryoEM)
From Stagg et al. Nature 439, 234, 2006
Liposomes were made by extrusion through a 400 nm filter.
Purified Sar1p was added with or without nucleotides.
Samples were fixed, and thin sections were examined with electron microscopy.
The results show that Sar1p + GTP causes membrane tubulation.
Fig. 2. Membrane Curvature Is Generated by the N-Terminal Helix of Sar1p.
Panel A: Sedimentation analysis of liposome binding by wt-Sar1p or by D23-Sar1p (a 6-histidine tag replacing the 23 residue N-terminal helix).
The proteins were incubated with GMP-PNP and normal liposomes or liposomes supplemented with DOGS-NTA-Ni (Ni-tagged lipids). The 6-histidine tag binds to Ni. Imidazole is a histidine-analog that prevents binding to Ni. Binding was determined by pelleting the liposomes.
Panel B: Tryptophan fluorescence as a measure of nucleotide binding.
Sec12 stimulates nucleotide exchange.
Sec23/24p (GAP) and Sec13/31p (coat) stimulate GTP hydrolysis.
Conclusion: The 6-his tag allows for liposome binding seemingly without affecting GTP exchange or hydrolysis.
Panel C: Ni-liposomes alone (top),
with GTP and wt-Sar1p (middle)
or D23-Sar1p (bottom).
Panel D: Surface density of tubules generated with wt-Sar1p (black bars) or D23-Sar1p (grey bars) and GTP (+).
Coclusion:D23-Sar1p can still tubulate membrae, but not as well as wt Sar1p
Panel A and B: wt Sar1p or D23-Sar1p and the COPII coat proteins sec23/24p and sec13/31p were added to Ni-liposomes.
Conclusion: D23-Sar1p can aid in bud formation, but there seem to be fewer tubules and severed vesicles.
Apparently sec23/24p and sec13/31p help form buds, even if D23-Sar1p alone is not very efficient (Fig. 2).
Panel C: Quantitation of the finding that more buds are formed with D23-Sar1p and COPII.
Panel D: This budding requires sec13/31.
Conclusion:D23-Sar1p form buds
with COPII, but this budding is due to recruitment of sec13/31.
Low, because vesicles are formed
Fig. 4. Direct Recruitment of Sec23/24p and Sec13/31p to Microsomes Does Not Bypass the Requirement for Full-Length Sar1p in Vesicle Budding
Microsomes, supplemented with Ni-lipids, were loaded with 35S-labeled glycosylated a-factor (gpaf) and stripped with urea. Budding reactions include a 6-his tagged version of sec23/24 and sec13/31, wt Sar1p or D23-Sar1p and nucleotide (nuc).
Panel A: Controls showing recruitment of sec24 and sec31 to the microsomes. Binding was monitored by sedimentation.
Panel B: Vesicles were separated from microsomes by differential centrifugation. D23-Sar1 can support vesicle formation but only at higher COPII concetrations.
Panel C: Recruitment of gpaf and sed5 (cargo molecules) into prebudding complexes is not affected by Sar1p or DSar1p.
Fig. 5. Mutations in the N-Terminal Helical Domain of Sar1p Reduce Membrane Tubulation
Panel A: Helical wheel showing residues that were targeted for mutation
Panel B: Flotation assay shows that some mutations in the N-terminal helix of Sar1 affect lipid binding, but not recruitment of other COPII proteins to the liposomes.
Panel C: The effects of N terminal mutations on liposome binding are reflected by the ability to tubulate membranes.
Panel D: Quantitation of the same.
Fig. 6. n-Sar1p Mutants Are Defective in Vesicle Budding despite Normal Recruitment of Coat and Cargo Proteins
Panel A: Microsomes were loaded with gpaf, incubated with Sar1 and COPII proteins and vesicle formation was assessed by differential centrifugation.
Conclusion: The effects of mutations on vesicle formation are similar to their effects on tubulation.
Panel B: The effects are strongest at lower concentrations of Sar1p.
Panel C: Recruitment of Sec23 and Sec24 to the microsomal membranes is not impaired by N terminal mutations.
Panel D: Formation of prebudding complexes is also not impaired.
Recruitment of gpaf and Sed5p in microsomes by sec24p was assessed by solubilizing the complex with digitonin and coIP with Ni-NTA using a 6-his tagged version of Sec23p.
Panel A: Yeast with mutant Sar1p grow well.
Panel B (below): Pulse chase experiment showing that Gas1p maturation is impaired. The precusor (p) is in the ER and the mature form (m) is in the Golgi.
Panel C (right): Quantitation of the pulse chase in panel B.
Pulse chase was done with a short pulse of in vivo labeling, followed by a chase without labe, lysis, immunoprecipitation and SDS PAGE
Fig. 8. The n1-Sar1p mutant does not support efficient membrane fission.
Panel A and B: Thin tubules and constrictions are formed when COPII proteins are added to wt Sar1p but not when they are aded to n1-Sar1p.
Panel C (below): Quantitation of the results in Panels A and B.
Sar1p-GTP is able to tubulate liposomes.
The N-terminal Helix of Sar1p is required for efficient tubulation.
Mutations in this helix disrupt vesicle formation despite normal recruitment of cargo molecules to COPII “prebuds”.
GTP bound Sar1p may initiate localized membrane deformations that are “captured” by electrostatic interactions between the lipid bilayer and Sec23/ 24p and propagated upon Sec13/31p recruitment.
Annu Rev Cell Dev Biol 20, 87-123, 2004
Maturation of Golgi cisternae Vesicular transport
Uses Retrograde vesicles Uses Anterograde vesicles
“Procollagen traverses the Golgi stack without leaving the lumen of cisternae: evidence for cisternal maturation.”
Examples of swollen Golgi cisternae:
A: 0 min with DPD , distensions are everywhere
B: 10 min with DPD , distensions are in trans and medial cisternae
C: 30 min with DPD, distensions are in the TGN
The arrows and inserts show clathrin coated pits which are in the TGN and therefore help determine the orientation of the Golgi stacks.
Two new papers retest the idea of Golgi maturation through live imaging of secreted proteins in yeast. (Nature 441, 1002-1006 and 1007-1010, 2006)