Chapter 6
Cytoplasmic matrix,Endomembrane system,
Protein Sorting and membrane trafficking
Learning objective
1,Compartmentalization in Eukaryotic Cells;
2,The structural and functional relationship among the ER,
Golgi complexes,lysosomes and plasma membranes of
eukaryotic cells;
3,The pathways of proteins targeting and sorting,and its
mechanisms;
4,The ways of protein modifications and intracellular sites
after they are synthesized;
5,Types of vesicle transport and their functions.
1,The Compartmentalization in Eukaryotic Cells
?Membranes divide the cytoplasm of eukaryotic cells into
distinct compartments.
Three categories in eukaryotic cells:
(1) the endomembrane system,ER,Golgi complex,Lys.,
secretory vesicles,
(2) the cytosol.
(3) mitochondria,chloroplasts,peroxisomes,and the nucleus,
?Membrane-bound structures (organelles) are found in all
eukaryotic cells.
?Cytoplasmic matrix and its functions
?Cytoplasmic Matrix,The region of fluid content
of the cytoplasm outside of the membranous
organelles,Aqueous solution of large and small
molecules including filaments of cytoskeleton which
act as organizer for some order.
?The Cytosol is the site of protein synthesis and
degradation or modification,It also performs most
of the cell’s intermediary metabolism.
A,Cytoplasmic matrix (Cytosol) and
Endomembrane System
Functions of cytoplasmic matrix:
The protein synthesis,degradation and modification.
Cells carefully monitor the amount of misfolded proteins,An
accumulation of misfolded proteins in the cytosol triggers a heat-
shock response,which stimulates the transcription of genes
encoding cytosolic chaperones that help to refold the proteins.
B,Endomembrane System
?Endomembrane System, The structural and functional relationship organelles including
ER,Golgi complex,lysosome,endosomes,secretory vesicles.
?Membrane-bound structures (organelles) are found in all eukaryotic cells.
Intracellular compartment % of total cell volume
Cytosol 54
Mittchondria 22
Rough ER cisternae 9
Smooth ER cisternae plus Golgi cisternae 6
Nucleus 6
Peroxisome 1
Lysosomes 1
Endosomes 1
Relative volumes occupied by the major
intracellular compartments in Liver Cell
C,The Dynamic Nature of the Endomembrane System
?Most organelles are part of a dynamic system in which
vesicles move between compartments.
?Biosynthetic parthways move proteins,carbohydrates
and lipids within the cell.
?Secretory pathways discharge proteins from cells.
?Endocytic parthways move materials into cells.
?Sorting signals are recognized by receptors and target
proteins to specific sites.
D,A few approaches to the study of cytomembranes
?Insights gained from autoradiography;
?Insights gained from the biochemical analysis of
subcellular fractions;
?Insights gained from the study of genetic mutants;
The dynamic activities of endomembrane systems
are highly conserved despite the structural
diversity of different cell types.
De Duve,A.Claude and G.Palade,1974 Nobel Plrize
2,The structure and functions of Endoplasmic
Reticulum(ER)
Rough endoplasmic reticulum and Smooth endoplasmic reticulum
RER has ribosomes on the cytosolic side of continuous,flattened sacs(cisternae);
SER is an interconnecting network of tubular membrane elements.
Microsome(100-200nm) rER of pancreatic cells
Microsomes are heterogeneous mixtures of similar-sized vesicles,formed from
membranes of the ER and Golgi complex,Microsomes retain activity during
purification,allowing studies of function and composition.
A,Functions of the
rER
? Proteins synthesized
on ribosomes of rER
include:
? secretory proteins,
? integral membrane
proteins,
? soluble proteins of
organelles.
? Modification and processing of newly synthesized
proteins,glycosylation in the rER;
N-linked,linked to the amide nitrogen of asparagine (ER)
O-linked,linked to the hydroxyl group serine or threonine via GalNac (in Golgi)
The precursor of 14
residues is the same
in plants,animals,
and single-celled
eukaryotes
then remove 3
glucoses and 1
mannose in the ER
?Quality control of of newly synthesized proteins---The
role of N-linked glycosylation in ER protein folding
Quality control,ensuring that misfolded proteins do not leave ER
The lumen of rER contains:
?Bip and calnexin (chaperones),
that recognize and bind to unfolded
or misfolded proteins and give them
correct conformation;
?Protein disulfide isomerase
( PDI ) ;
?GT(glucosyl-transferase,
monitoring enenzyme ) recognize
unfolded or misfolded proteins and
adds a glucose to the end of oligo.,
?Synthesis of membrane lipids
? Most membrane lipids are synthesized
enterly within the ER.
? There are two exceptions:
(1) sphingomyelin and glycolipids,
(begins in ER; completed in Golgi);
(2) some of the unique lipids of the Mit
and Chl membranes (themself).
? The membranes of different 0rganelles
have markedly different lipids
composition.
? Transport by budding,ER→GC, Ly,PM
? Transport by phospholipid exchange
proteins(PEP),ER→other organelles
（ including Mit and Chl）
The role of phospholipid translocators in lipid bilayer synthesis
phospholipid
translocators =
Scramblase
(ABC
transporter
Family)
B,Functions of the sER
?Synthesis of steroids in endocrine cells.
?Detoxification of organic compounds in liver cells.
System of oxygenases---cytochrome p450 family
?Release of glucose 6-phosphate in liver cells.
?Sequestration of Ca2+.
Ca2+-ATPase
3,The structure and functions of Golgi complex
A.The polarity of Golgi complex
a) Cis cisternae of Golgi complex,reduced osmium tetroxide(OsO4);
b) Reaction for enzyme mannosidase II,localized in the medial;
c) Reaction for enzyme nucleoside diphosphatase,localized in the trans cisternae.
?Regional differences in membrane composition
across the Golgi stack
B,The Functions of Golgi complex
?Glycosylation in
the Golgi complex
Golgi complex
plays a key role in
the assembly of the
carbohydrate
component of
glycoproteins and
glycolipids.
The core carbohydrate of N-linked oligosaccharides is
assembled in the rER.
Modifications to N-linked oligosaccharides are completed in the
Golgi complex.
O-linked oligosaccharides takes place in Golgi complex.
Structure of typical O- and N-linked oligosaccharides
Core Region
After R,Kornfeld
and S,Kornfeld,
1985,Annu,Rev,
Biochem,45:631
?What is the purpose of glycosylation?
N-linked glycosylation is prevalent in all eucaryotes,
but is absent from procaryotes.
?It don’t require a template,There is an important difference
between the construction of an oligosaccharide and the synthesis of
DNA,RNA,and protein.
?Important functions:
(1) One might suspect that they function to aid folding and the
transport process; for example,carbohydrate as a marker during protein
folding in ER and the use of carbohydrate-binding lectins in guiding ER-to-
Golgi transport.
(2) Limit the approach of other macromolecules to the protein surface,
more resistant to digestion by proteases.
(3) Regulatory roles in signaling through the cell-surface receptor
Notch,to allows these cells to respond selectively to activating stimuli.
?The Golgi
networks are
processing and
sorting stations
where proteins
are modified,
segregated and
then shipped in
different
directions.
?Golgi complex and cell’s secretion
Continual,unregulated
discharge of material
from the cells
The discharge of
products stored in
cytoplasmic granules,
in response to
appropriate stimuli.
?Vesivular transport within the Golgi apparatus:
Two views,
cisternal maturation model and vesicular transport model
Two possible models explaining the organization of the Golgi
complex and the transport from one cisterna to the next.
十十十
C,Golgi Biogenesis
Stages of Golgi growth and
division,Shown are thin section
electron micrographs of T,gondii
RH tachyzoites replicating by
endodyogeny in HFF cells,Cells
were placed in one of four
categories according to the
number and size of the Golgi,a,
single Golgi; b,single,elongated
Golgi; c,two Golgi; d,Golgi,often
more vesiculated,in
each nascent daughter cell,
delineated by the growing inner
membrane complex (IMC),a,
apicoplast; dg,dense granules;
er,ER; es,ER exit sites on the
outer flattened part of the nuclear
envelope; G,Golgi; m,
micronemes; mit,mitochondria; r,
rhoptries,Scale bar,0.5mm.
Stable expression of mammalian Golgi proteins,a,b,Overlaid immunofluorescence and
phase images of GRASP–YFP (a) and NAGTI–YFP (b) in stable,transgenic cell lines of
Toxoplasma gondii,c–h,Immunofluorescence images of a transgenic cell line
expressing both GRASP–CFP (green) and NAGTI–YFP (red) before (c–e) or after (f–h)
treatment with 5mg /ml BFA for 10 min at 37oC,Merged images are shown on the right,
Asterisks indicate a secreted form of NAGTI–YFP that accumulates in the
parasitophorous vacuole,Scale bars,5mm.
Immunoelectron microscopy of
transgenic parasites,a–c,
Cryosections of GRASP–YFP
(a,c) or NAGTI–YFP (b)
transgenic parasites,pretreated
for 2 h with 50mg/ml
cycloheximide,before being
fixed and immunolabelled for
YFP using polyclonal antibodies
against GFP followed by protein
A coupled to 5-nm gold
particles,Note the high density
of labelling restricted to Golgi
membranes,In c,GRASP–YFP
transgenic parasites were
treated with BFA (5mg/ml) for
30 min before immunolabelling,
Note the tubulo-vesicular
appearance of the Golgi caused
by loss of Golgi enzymes to the
ER,d,Quantification of images
in a and b,Results are
presented as mean ± s.d,gold
particles /um2.
Biogenesis of the Golgi apparatus in living parasites,a–h,Transgenic parasites stably
expressing IMC1–CFP (blue) were transfected with plasmid DNA encoding GRASP–
YFP (green),After 20 h of infection in HFFs,four parasites were imaged by time-lapse
video fluorescence microscopy,Images were taken every 10 min for 7 h at 37 ° C,
Representative images at the indicated times are shown,Note that T,gondii,Replicates
synchronously in a given vacuole,which permits simultaneous imaging of several cells
at the same cell-cycle stage,i,j,Transgenic parasites expressing NAGTI–YFP (green)
were imaged over time and sample images late in cell division are shown,For both Golgi
markers note the inheritance of two structures by each nascent daughter (f,i,j) and their
eventual coalescence (arrow in g and h),k,Threedimensional reconstruction of two
parasites during mitosis,The Golgi was selectively outlined in red and other electron-
dense structures were coloured in green or dark blue to differentiate the two forming
daughter cells,Golgi are inherited by both cells,and in the complete reconstruction of
one daughter (right) two Golgi structures are visible (arrows),Note that the other
daughter was only reconstructed partially and contains a single Golgi structure.
4,The structure and functions of Lysosomes
A,Characteristics of Lysosomes
① Lysosome is a heterogenous organelle,
Primary lysosomes
Second lysosomes
heterophagic
autophagic
Residual body
Primary Lys.
Second Lys
Figure 6-19 Histochemical visualization
of lysosomes,Electron micro-graphs
of two sections of a cell stained to reveal
the location of acid phosphatase,a
marker enzyme for lysosomes,The larger
membrane-bounded organelles,
containing dense precipitates of lead
phosphate,are lysosomes,whose
diverse morphology reflects variations in
the amount and nature of the material
they are digesting,The precipitates are
produced when tissue fixed with
glutaraldehyde is incubated with a
phosphatase substrate in the presence of
lead ions,Two small vesicles thought to
be carrying acid hydrolases from the
Golgi apparatus are indicated by red
arrows in the top panel,(Courtesy of
Daniel S,Friend.)
② Lysosomes contain plenty acid hydrolases that
can digest every kind of biological molecule,
---the principal sites of intracellular digestion.
Marker enzyme,acid phosphatase
③ Lysosome membrane,
H+-pumps,internal proton
concentration is kept high by H+-
ATPase
Glycosylated proteins,may
protect the lysosome from self-
digestion.
Transport proteins,
transporting digested materials.
Figure 13-18 The low pH in lysosomes and endosomes,Proteins labeled
with a pH-sensitive fluorescent probe (fluorescein) and then endocytosed by
cells can be used to measure the pH in endosomes and lysosomes,The
different colors reflect the pH that the fluorescent probe encounters in these
organelles,The pH in lysosomes (red) is about 5,while the pH in various types
of endosomes (blue and green) ranges from 5.5 to 6.5,(Courtesy of Fred
Maxfield and Kenneth Dunn.)
Figure 13-20 The plant cell vacuole,This electron micrograph of cells in a
young tobacco leaf shows that the cytosol is confined by the enormous vacuole
to a thin layer,containing chloroplasts,pressed against the cell wall,The
membrane of the vacuole is called the tonoplast,(Courtesy of J,Burgess.)
B,The Functions of Lysosomes
?Lysosomes are involved in three major cell functions:
① phagocytosis; ② autophagy; ③ endocytosis.
Primary lys fuse with either phagocytic or autophagic vesicles,forming
residual bodies that either undergo exocytosis or are retained in the cell as
lipofuscin granules.
C,Lysosomes and Diseases
Disorders resulting from defects in lysosomal function:
① Autolysis,A break or leak in the membrane of lys releases
digestive enzymes into the cell which damages the surrounding
tissues (Silicosis).
② Lysosomal storage diseases are due to the absence of one or
more lysosomal enzymes,and resulting in accumulation of
material in lysosomes as large inclusions.
One severe type of the disease is I-cell disease (inclusion –
cell disease,GlcNAc-Phosphotransferase gene mutant).
Tay-Sachs disease results from a deficiency of the enzyme (-
N-hexosaminidase A) whose function is to degrade gangliosides,
a major component of brain cell membranes.
表 1,神经鞘脂 贮 积病
疾病 缺失酶类 主要贮积底物 后果
GM1神经节苷脂贮
积症
GM1?-半乳糖苷
酶
神 经 节 苷 脂
GM1
智力迟钝, 肝脏肥大, 骨
骼受累, 2岁前死亡
泰－萨二氏病 己糖胺酶 A 神 经 节 苷 脂
GM2
智力迟钝, 失明, 3岁前死
亡
法布莱氏病 ?-半乳糖苷酶 A 三己糖神经酰
胺
皮疹, 肾功能丧失, 下肢
疼痛
山霍夫氏病 己糖胺酶 A和 B 神 经 节 苷 脂
GM2 和红细胞
糖苷酯
与泰－萨氏疾病症状相似,
但发展更快
高歇氏病 葡糖脑苷酯酶 葡糖脑苷脂 肝脏和脾脏肿大, 长骨腐
蚀, 只在婴儿期发生智力
迟钝
尼 -皮二氏病 鞘磷脂水解酶 鞘磷脂 肝脏和脾脏肿大, 智力迟
钝
Farber’s 脂肪肉芽肿
病
神经酰胺水解酶 神经酰胺 疼痛性与退行性的关节变
形, 皮肤瘤, 几年内死亡
Krabbe’s 病 半乳糖脑苷酯酶 半乳糖脑苷脂 髓磷脂缺失, 智力迟钝, 2
岁前死亡
脑硫脂沉积 芳基硫酸酯酶 脑硫脂 智力迟钝, 前十年死亡
D,Biogenesis of Lysosomes
Figure 6-23 The transport of newly synthesized lysosomal hydrolases to lysosomes,The
precursors of lysosomal hydrolases are covalently modified by the addition of mannose 6-phosphate in the CGN,
They then become segregated from all other types of proteins in the TGN because a specific class of transport
vesicles budding from the TGN concentrates mannose 6-phosphate-specific receptors,which bind the modified
lysosomal hydrolases,These vesicles subsequently fuse with late endosomes,At the low pH of the late endosome
the hydrolases dissociate from the receptors,which are recycled to the Golgi apparatus for further rounds of
transport,In late endosomes the phosphate is removed from the mannose on the hydrolases,further ensuring that
the hydrolases do not return to the Golgi apparatus with the receptor,
Mannose 6-phosphate residues target proteins
to lysosomes
Targeting of soluble
lysosomal enzymes
to endosomes and
lysosomes by M-6-P
tag
Phosphorylation of mannose residues on
lysosomal enzymes catalyzed by two enzymes
Recognition
site binds to
Signal patch
GlcNAc
phosphotransferase
phosphodiesterase
Figure 6-40,The mannose 6-phosphate (M6P) pathway,the major route for targeting
lysosomal enzymes to lysosomes,Precursors of lysosomal enzymes migrate from the rER to the cis-Golgi where
mannose residues are phosphorylated,In the TGN,the phosphorylated enzymes bind to M6P receptors,which direct the
enzymes into vesicles coated with the clathrin,The clathrin lattice surrounding these vesicles is rapidly depolymerized to its
subunits,and the uncoated transport vesicles fuse with late endosomes,Within this low-pH compartment,the phosphorylated
enzymes dissociate from the M6P receptors and then are dephosphorylated,The receptors recycle back to the Golgi,and the
enzymes are incorporated into a different transport vesicle that buds from the late endosome and soon fuses with a lysosome,
The sorting of lysosomal enzymes from secretory proteins thus occurs in the TGN,and these two classes of proteins are
incorporated into different vesicles,which take different routes after they bud from the Golgi.[G,Griffiths et al.,Cell 52:329;
S,Kornfeld,Annu,Rev,Biochem,61:307; and G,Griffiths and J,Gruenberg,Trends Cell Biol,1:5]
5,Protein Sorting
A,Overview of sorting of nuclear-encoded
proteins in eukaryotic cells
?Proteins are imported into organelles by three
mechanisms:
?Gated Transport,Transport through nuclear pores
?Transmembrane transport,ER,Mit,Chl,Per
?Vesicular transport,ER-Golgi-PM-Lys,Endosome
Road map of protein sorting
?Protein sorting,Protein molecules move from the
cytosol to their target organelles or cell surface
directed by the sorting signals in the proteins.
?Signal peptides and Signal patches
Figure 6-8 Two ways that a sorting signal can be built into a protein.(A) The
signal resides in a single discrete stretch of amino acid sequence,called a signal
peptide,that is exposed in the folded protein,Signal peptides often occur at the end of
the polypeptide chain,but they can also be located elsewhere,(B) A signal patch can be
formed by the juxtaposition of amino acids from regions that are physically separated
before the protein folds; alternatively,separate patches on the surface of the folded
protein that are spaced a fixed distance apart could form the signal,
Gated transport:
Through gated pores—Nuclear pores;
Nuclear localization signal (NLS);
Folded and assembly form to transport.
Transmembrane transport
ER signal sequence,
Mit,Chl,Per,Leader sequence;
Through translocon on the membrane;
Single and Unfold form;
Helped by molecular chaperons
Vesicular transport
Budding,transporting,docking and at last
fusion with target membrane;
Assembly coated proteins on the vesicles
(Clathrin,COPII and COPI);
Only Properly folded and assembled proteins;
The orientation of transported proteins and
lipids is not changed during transporting.
B,Signal Hypothesis
--G.Blobel & D.Sabatini,1971.
A model for the Signal Mechanism
of Cotranslational Import
Evidence That Protein
Synthesized on Ribosomes
Attached to ER Membranes
Pass Directly into the ER
Lumen
(D.Sabatini)
Milstein, IgG
?Milstein et al,Studying the synthesis of light chain of IgG (in
cell-free systems,20 Aa longer at N-terminal end than the
authentic light chain )
Adding ER membranes
to this system leads to
the production of an IgG
light chain of the correct
size.
?A Schematic model for the synthesis of a
secretory protein on a membrane-bound
ribosome of the rough ER
P6 99
?Signal-recognition particle,SRP,Six different polypeptides
complexed with a 300-nucleotide (7S)molecule of RNA.
?ER signal sequence,Typically 15-30 amino acids:
Consist of three domains,a positively charged N-terminal region,a
central hydrophobic region,and a polar region adjoining the site where
cleavage from the mature protein will take place,A signal sequence on
nascent seretory proteins targets them to the ER and is then cleaved off
?SRP receptor (GTP binding protein)
SRP have three main active sites:
One that recognizes and binds to
ER signal sequence;
One that interacts with the
ribosome to block further
translation;
One that binds to the ER
membrane (docking protein))
The sorting signal
of VSV
glycoproteins,
Asp-X-Gln或 DXE
Figure 6-43,The sorting of proteins destined for the apical and basolateral plasma
membranes of epithelial cells,When cultured MDCK cells are infected simultaneously with
VSV and influenza virus,the VSV glycoprotein is found only on the basolateral membrane,
whereas the HA glycoprotein of the influenza virus is found only on the apical membrane,Like
these viral proteins,some cellular proteins are sorted directly to the apical membrane and others
to the basolateral membrane via specific transport vesicles that bud from the trans-Golgi network,
In certain other polarized cells,some apical and basolateral proteins are transported together to
the basolateral surface; the apical proteins then move selectively,by endocytosis and transcytosis,
to the apical membrane,[K,Simons et al.,Cell 62:207; K,Mostov et al.,JCB,116:577]
?Start-transfer Sequence &
Stop-transfer Sequence
Figure 6-24,Synthesis and insertion into the ER membrane of the GLUT1 glucose
transporter and other proteins with multiple transmembrane ?-helical segments.
The N-terminal ? helix functions as an internal,uncleaved signal-anchor sequence (red),directing binding of
the nascent polypeptide chain to the rER membrane and initiating cotranslational insertion,Both SRP and the
SRP receptor are involved in this step,Following synthesis of helix 2,which functions as a stop-transfer
membrane-anchor sequence,extrusion of the chain through the translocon into the ER lumen ceases,The first
two ? helices then move out of the translocon into the ER bilayer,anchoring the nascent chain as an ?-helical
hairpin,The C-terminus of the nascent chain continues to grow in the cytosol,Subsequent ?-helical hairpins
could insert similarly,although SRP and the SRP receptor are required only for insertion of the first
signalanchor sequence,Although only six transmembrane ? helices are depicted here,GLUT1 and proteins
of similar structure have twelve or more,[H,P,Wessels et al.,Cell 55:61.]
?The Orientation of Nascent Polypeptide
The Nascent polypeptide is oriented within translocon
so that the positively charged flanking sequence faces the
cytosol
C,A Model for the Postranslational Import
of Polypeptides into the Mit.
?Post-translational modification and quality control
in the rER
Disulfide bonds are formed and
rearranged in the ER lumen
Only in ER lumen is there a redox
environment for oxidation of –SH
groups.
PDI,protein disulfide
isomerase,found in abundance
in the ER lumen
?Correct
folding of newly
made proteins is
facilitated by
several ER
proteins
Proteins without
any signal
sequence are
cytosol residual
proteins
6,Types of Vesicle Transport and Their Functions
A,The three different types of coated vesicles.
Different coat proteins select different cargo and shape the
transport vesicles that mediate the various steps in the
biosynthetic-secretory and endocytic pathways.
?COPII-coated vesicles move materials from the ER
to the Golgi.
The assembly of a COPII-coated vesicles.
Sar—GTP binding protein,Sar-GTP binds to the ER; Sar-
GDP dissociates from the ER
Antibodies is
able to block the
budding of
vesicle from ER
but have no
effect on vesicle
transport from
one Golgi
compartment to
another in
mammalian cell.
? COPI-coated vasicles transporting Escaped ER
resident Proteins Back to the ER.
(1) The assembly of a COPI-coat is mediated by ADP-ribosylation
factor(ARF),GTP binding protein,which is required for vesicle
transfer between cisternae.
(2) COPI coated vesicles may select specific cargo.
?ER is an open prison,?Soluble ER protein bear Retrieving signal—
KDEL(Lys-Asp-Glu-Leu)in mammal and HDEL in yeast,whereas ER
membrane proteins bear the signal KKXX,?The KDEL receptor
present in vesicular tubular clusters and the Golgi apparatus..
(3) COPI-coated vesicle were first identified by treatment of GTP
analogues --- COPI-coated vesicle accumulated within the cell
and could be isolated by centrifugation.
A model for the retrieval of ER resident proteins.
The KDEL receptor captures the soluble ER resident proteins and
carries them in COPI-coated transport vesicles back to the ER.
Neutral pH,dissociate from the KDEL; low pH,binding the KDEL
? Clathrin-coated vesicle,Transporting Cargo from
the TGN to endosomes,Lysosomes,and plant
vacuoles and also move materials from the PM to
cytoplasmic compartments along the endocytic
pathway.
(1) The TGN of Golgi is the Sourse of Clathrin-coated vesicle.
(2) Clathrin-coats contain,
protein clathrin-----which forms a structural scaffold,
adaptors---- multisubunit,which forms an inner shell.
The formation of clathrin-coated
pits in the TGN
B,The SNARE Hypothesis for Transport
Vesicle Targeting and Fusion
?Specificity in vesicle
docking and fusion is thought
to be attained through
specific interactions between
specific v-SNARE proteins
on the vesicle membranes
and t-SNARE proteins on the
membranes of the target
compartment.
?SNAREs are a protein
family,There are at least 20
different SNAREs in an
animal cell.The role of SNAREs in guiding
vesicular transport.
The struction of paired SNAREs (the four-helix bundle).
The SNAREs responsible for docking synaptic vesicles at
the plasma membrane of nerve terminals consist of three
proteins.
The basic molecular components in eukaryotic cells include
v-SNAREs (v-SNAP receptors) on transport vesicles,t-SNAREs
(t-SNAP receptors) on target membranes,Rab GTPase,NSF,and
several SNAPs,SNAPs are soluble NSF attachment proteins.
The role of Rab proteins in
facilitating the docking of
transport vesicles.
GEF in donor membrane;
GTP binding alters the
conformation of Rab,exposing
its covalently attached lipid
group and helps anchoring;
Rab and Rab effectors help the
vesicle dock and pairing of the v-
and t-SNAREs;
GDI,GDP dissociation inhibitor.
kinesin dynein
C,Motor proteins mediate movement of
vesicles along MT
A General Model for Kinesin-and Dynein-
mediated Transport in a Typical Cell
D,Maintenance of membrane asymmetry
E,Membrane flow
作 业
? 膜泡表面标志与膜泡运输
? 过氧化物酶体中蛋白质的
来源及输入机制
Cytoplasmic matrix,Endomembrane system,
Protein Sorting and membrane trafficking
Learning objective
1,Compartmentalization in Eukaryotic Cells;
2,The structural and functional relationship among the ER,
Golgi complexes,lysosomes and plasma membranes of
eukaryotic cells;
3,The pathways of proteins targeting and sorting,and its
mechanisms;
4,The ways of protein modifications and intracellular sites
after they are synthesized;
5,Types of vesicle transport and their functions.
1,The Compartmentalization in Eukaryotic Cells
?Membranes divide the cytoplasm of eukaryotic cells into
distinct compartments.
Three categories in eukaryotic cells:
(1) the endomembrane system,ER,Golgi complex,Lys.,
secretory vesicles,
(2) the cytosol.
(3) mitochondria,chloroplasts,peroxisomes,and the nucleus,
?Membrane-bound structures (organelles) are found in all
eukaryotic cells.
?Cytoplasmic matrix and its functions
?Cytoplasmic Matrix,The region of fluid content
of the cytoplasm outside of the membranous
organelles,Aqueous solution of large and small
molecules including filaments of cytoskeleton which
act as organizer for some order.
?The Cytosol is the site of protein synthesis and
degradation or modification,It also performs most
of the cell’s intermediary metabolism.
A,Cytoplasmic matrix (Cytosol) and
Endomembrane System
Functions of cytoplasmic matrix:
The protein synthesis,degradation and modification.
Cells carefully monitor the amount of misfolded proteins,An
accumulation of misfolded proteins in the cytosol triggers a heat-
shock response,which stimulates the transcription of genes
encoding cytosolic chaperones that help to refold the proteins.
B,Endomembrane System
?Endomembrane System, The structural and functional relationship organelles including
ER,Golgi complex,lysosome,endosomes,secretory vesicles.
?Membrane-bound structures (organelles) are found in all eukaryotic cells.
Intracellular compartment % of total cell volume
Cytosol 54
Mittchondria 22
Rough ER cisternae 9
Smooth ER cisternae plus Golgi cisternae 6
Nucleus 6
Peroxisome 1
Lysosomes 1
Endosomes 1
Relative volumes occupied by the major
intracellular compartments in Liver Cell
C,The Dynamic Nature of the Endomembrane System
?Most organelles are part of a dynamic system in which
vesicles move between compartments.
?Biosynthetic parthways move proteins,carbohydrates
and lipids within the cell.
?Secretory pathways discharge proteins from cells.
?Endocytic parthways move materials into cells.
?Sorting signals are recognized by receptors and target
proteins to specific sites.
D,A few approaches to the study of cytomembranes
?Insights gained from autoradiography;
?Insights gained from the biochemical analysis of
subcellular fractions;
?Insights gained from the study of genetic mutants;
The dynamic activities of endomembrane systems
are highly conserved despite the structural
diversity of different cell types.
De Duve,A.Claude and G.Palade,1974 Nobel Plrize
2,The structure and functions of Endoplasmic
Reticulum(ER)
Rough endoplasmic reticulum and Smooth endoplasmic reticulum
RER has ribosomes on the cytosolic side of continuous,flattened sacs(cisternae);
SER is an interconnecting network of tubular membrane elements.
Microsome(100-200nm) rER of pancreatic cells
Microsomes are heterogeneous mixtures of similar-sized vesicles,formed from
membranes of the ER and Golgi complex,Microsomes retain activity during
purification,allowing studies of function and composition.
A,Functions of the
rER
? Proteins synthesized
on ribosomes of rER
include:
? secretory proteins,
? integral membrane
proteins,
? soluble proteins of
organelles.
? Modification and processing of newly synthesized
proteins,glycosylation in the rER;
N-linked,linked to the amide nitrogen of asparagine (ER)
O-linked,linked to the hydroxyl group serine or threonine via GalNac (in Golgi)
The precursor of 14
residues is the same
in plants,animals,
and single-celled
eukaryotes
then remove 3
glucoses and 1
mannose in the ER
?Quality control of of newly synthesized proteins---The
role of N-linked glycosylation in ER protein folding
Quality control,ensuring that misfolded proteins do not leave ER
The lumen of rER contains:
?Bip and calnexin (chaperones),
that recognize and bind to unfolded
or misfolded proteins and give them
correct conformation;
?Protein disulfide isomerase
( PDI ) ;
?GT(glucosyl-transferase,
monitoring enenzyme ) recognize
unfolded or misfolded proteins and
adds a glucose to the end of oligo.,
?Synthesis of membrane lipids
? Most membrane lipids are synthesized
enterly within the ER.
? There are two exceptions:
(1) sphingomyelin and glycolipids,
(begins in ER; completed in Golgi);
(2) some of the unique lipids of the Mit
and Chl membranes (themself).
? The membranes of different 0rganelles
have markedly different lipids
composition.
? Transport by budding,ER→GC, Ly,PM
? Transport by phospholipid exchange
proteins(PEP),ER→other organelles
（ including Mit and Chl）
The role of phospholipid translocators in lipid bilayer synthesis
phospholipid
translocators =
Scramblase
(ABC
transporter
Family)
B,Functions of the sER
?Synthesis of steroids in endocrine cells.
?Detoxification of organic compounds in liver cells.
System of oxygenases---cytochrome p450 family
?Release of glucose 6-phosphate in liver cells.
?Sequestration of Ca2+.
Ca2+-ATPase
3,The structure and functions of Golgi complex
A.The polarity of Golgi complex
a) Cis cisternae of Golgi complex,reduced osmium tetroxide(OsO4);
b) Reaction for enzyme mannosidase II,localized in the medial;
c) Reaction for enzyme nucleoside diphosphatase,localized in the trans cisternae.
?Regional differences in membrane composition
across the Golgi stack
B,The Functions of Golgi complex
?Glycosylation in
the Golgi complex
Golgi complex
plays a key role in
the assembly of the
carbohydrate
component of
glycoproteins and
glycolipids.
The core carbohydrate of N-linked oligosaccharides is
assembled in the rER.
Modifications to N-linked oligosaccharides are completed in the
Golgi complex.
O-linked oligosaccharides takes place in Golgi complex.
Structure of typical O- and N-linked oligosaccharides
Core Region
After R,Kornfeld
and S,Kornfeld,
1985,Annu,Rev,
Biochem,45:631
?What is the purpose of glycosylation?
N-linked glycosylation is prevalent in all eucaryotes,
but is absent from procaryotes.
?It don’t require a template,There is an important difference
between the construction of an oligosaccharide and the synthesis of
DNA,RNA,and protein.
?Important functions:
(1) One might suspect that they function to aid folding and the
transport process; for example,carbohydrate as a marker during protein
folding in ER and the use of carbohydrate-binding lectins in guiding ER-to-
Golgi transport.
(2) Limit the approach of other macromolecules to the protein surface,
more resistant to digestion by proteases.
(3) Regulatory roles in signaling through the cell-surface receptor
Notch,to allows these cells to respond selectively to activating stimuli.
?The Golgi
networks are
processing and
sorting stations
where proteins
are modified,
segregated and
then shipped in
different
directions.
?Golgi complex and cell’s secretion
Continual,unregulated
discharge of material
from the cells
The discharge of
products stored in
cytoplasmic granules,
in response to
appropriate stimuli.
?Vesivular transport within the Golgi apparatus:
Two views,
cisternal maturation model and vesicular transport model
Two possible models explaining the organization of the Golgi
complex and the transport from one cisterna to the next.
十十十
C,Golgi Biogenesis
Stages of Golgi growth and
division,Shown are thin section
electron micrographs of T,gondii
RH tachyzoites replicating by
endodyogeny in HFF cells,Cells
were placed in one of four
categories according to the
number and size of the Golgi,a,
single Golgi; b,single,elongated
Golgi; c,two Golgi; d,Golgi,often
more vesiculated,in
each nascent daughter cell,
delineated by the growing inner
membrane complex (IMC),a,
apicoplast; dg,dense granules;
er,ER; es,ER exit sites on the
outer flattened part of the nuclear
envelope; G,Golgi; m,
micronemes; mit,mitochondria; r,
rhoptries,Scale bar,0.5mm.
Stable expression of mammalian Golgi proteins,a,b,Overlaid immunofluorescence and
phase images of GRASP–YFP (a) and NAGTI–YFP (b) in stable,transgenic cell lines of
Toxoplasma gondii,c–h,Immunofluorescence images of a transgenic cell line
expressing both GRASP–CFP (green) and NAGTI–YFP (red) before (c–e) or after (f–h)
treatment with 5mg /ml BFA for 10 min at 37oC,Merged images are shown on the right,
Asterisks indicate a secreted form of NAGTI–YFP that accumulates in the
parasitophorous vacuole,Scale bars,5mm.
Immunoelectron microscopy of
transgenic parasites,a–c,
Cryosections of GRASP–YFP
(a,c) or NAGTI–YFP (b)
transgenic parasites,pretreated
for 2 h with 50mg/ml
cycloheximide,before being
fixed and immunolabelled for
YFP using polyclonal antibodies
against GFP followed by protein
A coupled to 5-nm gold
particles,Note the high density
of labelling restricted to Golgi
membranes,In c,GRASP–YFP
transgenic parasites were
treated with BFA (5mg/ml) for
30 min before immunolabelling,
Note the tubulo-vesicular
appearance of the Golgi caused
by loss of Golgi enzymes to the
ER,d,Quantification of images
in a and b,Results are
presented as mean ± s.d,gold
particles /um2.
Biogenesis of the Golgi apparatus in living parasites,a–h,Transgenic parasites stably
expressing IMC1–CFP (blue) were transfected with plasmid DNA encoding GRASP–
YFP (green),After 20 h of infection in HFFs,four parasites were imaged by time-lapse
video fluorescence microscopy,Images were taken every 10 min for 7 h at 37 ° C,
Representative images at the indicated times are shown,Note that T,gondii,Replicates
synchronously in a given vacuole,which permits simultaneous imaging of several cells
at the same cell-cycle stage,i,j,Transgenic parasites expressing NAGTI–YFP (green)
were imaged over time and sample images late in cell division are shown,For both Golgi
markers note the inheritance of two structures by each nascent daughter (f,i,j) and their
eventual coalescence (arrow in g and h),k,Threedimensional reconstruction of two
parasites during mitosis,The Golgi was selectively outlined in red and other electron-
dense structures were coloured in green or dark blue to differentiate the two forming
daughter cells,Golgi are inherited by both cells,and in the complete reconstruction of
one daughter (right) two Golgi structures are visible (arrows),Note that the other
daughter was only reconstructed partially and contains a single Golgi structure.
4,The structure and functions of Lysosomes
A,Characteristics of Lysosomes
① Lysosome is a heterogenous organelle,
Primary lysosomes
Second lysosomes
heterophagic
autophagic
Residual body
Primary Lys.
Second Lys
Figure 6-19 Histochemical visualization
of lysosomes,Electron micro-graphs
of two sections of a cell stained to reveal
the location of acid phosphatase,a
marker enzyme for lysosomes,The larger
membrane-bounded organelles,
containing dense precipitates of lead
phosphate,are lysosomes,whose
diverse morphology reflects variations in
the amount and nature of the material
they are digesting,The precipitates are
produced when tissue fixed with
glutaraldehyde is incubated with a
phosphatase substrate in the presence of
lead ions,Two small vesicles thought to
be carrying acid hydrolases from the
Golgi apparatus are indicated by red
arrows in the top panel,(Courtesy of
Daniel S,Friend.)
② Lysosomes contain plenty acid hydrolases that
can digest every kind of biological molecule,
---the principal sites of intracellular digestion.
Marker enzyme,acid phosphatase
③ Lysosome membrane,
H+-pumps,internal proton
concentration is kept high by H+-
ATPase
Glycosylated proteins,may
protect the lysosome from self-
digestion.
Transport proteins,
transporting digested materials.
Figure 13-18 The low pH in lysosomes and endosomes,Proteins labeled
with a pH-sensitive fluorescent probe (fluorescein) and then endocytosed by
cells can be used to measure the pH in endosomes and lysosomes,The
different colors reflect the pH that the fluorescent probe encounters in these
organelles,The pH in lysosomes (red) is about 5,while the pH in various types
of endosomes (blue and green) ranges from 5.5 to 6.5,(Courtesy of Fred
Maxfield and Kenneth Dunn.)
Figure 13-20 The plant cell vacuole,This electron micrograph of cells in a
young tobacco leaf shows that the cytosol is confined by the enormous vacuole
to a thin layer,containing chloroplasts,pressed against the cell wall,The
membrane of the vacuole is called the tonoplast,(Courtesy of J,Burgess.)
B,The Functions of Lysosomes
?Lysosomes are involved in three major cell functions:
① phagocytosis; ② autophagy; ③ endocytosis.
Primary lys fuse with either phagocytic or autophagic vesicles,forming
residual bodies that either undergo exocytosis or are retained in the cell as
lipofuscin granules.
C,Lysosomes and Diseases
Disorders resulting from defects in lysosomal function:
① Autolysis,A break or leak in the membrane of lys releases
digestive enzymes into the cell which damages the surrounding
tissues (Silicosis).
② Lysosomal storage diseases are due to the absence of one or
more lysosomal enzymes,and resulting in accumulation of
material in lysosomes as large inclusions.
One severe type of the disease is I-cell disease (inclusion –
cell disease,GlcNAc-Phosphotransferase gene mutant).
Tay-Sachs disease results from a deficiency of the enzyme (-
N-hexosaminidase A) whose function is to degrade gangliosides,
a major component of brain cell membranes.
表 1,神经鞘脂 贮 积病
疾病 缺失酶类 主要贮积底物 后果
GM1神经节苷脂贮
积症
GM1?-半乳糖苷
酶
神 经 节 苷 脂
GM1
智力迟钝, 肝脏肥大, 骨
骼受累, 2岁前死亡
泰－萨二氏病 己糖胺酶 A 神 经 节 苷 脂
GM2
智力迟钝, 失明, 3岁前死
亡
法布莱氏病 ?-半乳糖苷酶 A 三己糖神经酰
胺
皮疹, 肾功能丧失, 下肢
疼痛
山霍夫氏病 己糖胺酶 A和 B 神 经 节 苷 脂
GM2 和红细胞
糖苷酯
与泰－萨氏疾病症状相似,
但发展更快
高歇氏病 葡糖脑苷酯酶 葡糖脑苷脂 肝脏和脾脏肿大, 长骨腐
蚀, 只在婴儿期发生智力
迟钝
尼 -皮二氏病 鞘磷脂水解酶 鞘磷脂 肝脏和脾脏肿大, 智力迟
钝
Farber’s 脂肪肉芽肿
病
神经酰胺水解酶 神经酰胺 疼痛性与退行性的关节变
形, 皮肤瘤, 几年内死亡
Krabbe’s 病 半乳糖脑苷酯酶 半乳糖脑苷脂 髓磷脂缺失, 智力迟钝, 2
岁前死亡
脑硫脂沉积 芳基硫酸酯酶 脑硫脂 智力迟钝, 前十年死亡
D,Biogenesis of Lysosomes
Figure 6-23 The transport of newly synthesized lysosomal hydrolases to lysosomes,The
precursors of lysosomal hydrolases are covalently modified by the addition of mannose 6-phosphate in the CGN,
They then become segregated from all other types of proteins in the TGN because a specific class of transport
vesicles budding from the TGN concentrates mannose 6-phosphate-specific receptors,which bind the modified
lysosomal hydrolases,These vesicles subsequently fuse with late endosomes,At the low pH of the late endosome
the hydrolases dissociate from the receptors,which are recycled to the Golgi apparatus for further rounds of
transport,In late endosomes the phosphate is removed from the mannose on the hydrolases,further ensuring that
the hydrolases do not return to the Golgi apparatus with the receptor,
Mannose 6-phosphate residues target proteins
to lysosomes
Targeting of soluble
lysosomal enzymes
to endosomes and
lysosomes by M-6-P
tag
Phosphorylation of mannose residues on
lysosomal enzymes catalyzed by two enzymes
Recognition
site binds to
Signal patch
GlcNAc
phosphotransferase
phosphodiesterase
Figure 6-40,The mannose 6-phosphate (M6P) pathway,the major route for targeting
lysosomal enzymes to lysosomes,Precursors of lysosomal enzymes migrate from the rER to the cis-Golgi where
mannose residues are phosphorylated,In the TGN,the phosphorylated enzymes bind to M6P receptors,which direct the
enzymes into vesicles coated with the clathrin,The clathrin lattice surrounding these vesicles is rapidly depolymerized to its
subunits,and the uncoated transport vesicles fuse with late endosomes,Within this low-pH compartment,the phosphorylated
enzymes dissociate from the M6P receptors and then are dephosphorylated,The receptors recycle back to the Golgi,and the
enzymes are incorporated into a different transport vesicle that buds from the late endosome and soon fuses with a lysosome,
The sorting of lysosomal enzymes from secretory proteins thus occurs in the TGN,and these two classes of proteins are
incorporated into different vesicles,which take different routes after they bud from the Golgi.[G,Griffiths et al.,Cell 52:329;
S,Kornfeld,Annu,Rev,Biochem,61:307; and G,Griffiths and J,Gruenberg,Trends Cell Biol,1:5]
5,Protein Sorting
A,Overview of sorting of nuclear-encoded
proteins in eukaryotic cells
?Proteins are imported into organelles by three
mechanisms:
?Gated Transport,Transport through nuclear pores
?Transmembrane transport,ER,Mit,Chl,Per
?Vesicular transport,ER-Golgi-PM-Lys,Endosome
Road map of protein sorting
?Protein sorting,Protein molecules move from the
cytosol to their target organelles or cell surface
directed by the sorting signals in the proteins.
?Signal peptides and Signal patches
Figure 6-8 Two ways that a sorting signal can be built into a protein.(A) The
signal resides in a single discrete stretch of amino acid sequence,called a signal
peptide,that is exposed in the folded protein,Signal peptides often occur at the end of
the polypeptide chain,but they can also be located elsewhere,(B) A signal patch can be
formed by the juxtaposition of amino acids from regions that are physically separated
before the protein folds; alternatively,separate patches on the surface of the folded
protein that are spaced a fixed distance apart could form the signal,
Gated transport:
Through gated pores—Nuclear pores;
Nuclear localization signal (NLS);
Folded and assembly form to transport.
Transmembrane transport
ER signal sequence,
Mit,Chl,Per,Leader sequence;
Through translocon on the membrane;
Single and Unfold form;
Helped by molecular chaperons
Vesicular transport
Budding,transporting,docking and at last
fusion with target membrane;
Assembly coated proteins on the vesicles
(Clathrin,COPII and COPI);
Only Properly folded and assembled proteins;
The orientation of transported proteins and
lipids is not changed during transporting.
B,Signal Hypothesis
--G.Blobel & D.Sabatini,1971.
A model for the Signal Mechanism
of Cotranslational Import
Evidence That Protein
Synthesized on Ribosomes
Attached to ER Membranes
Pass Directly into the ER
Lumen
(D.Sabatini)
Milstein, IgG
?Milstein et al,Studying the synthesis of light chain of IgG (in
cell-free systems,20 Aa longer at N-terminal end than the
authentic light chain )
Adding ER membranes
to this system leads to
the production of an IgG
light chain of the correct
size.
?A Schematic model for the synthesis of a
secretory protein on a membrane-bound
ribosome of the rough ER
P6 99
?Signal-recognition particle,SRP,Six different polypeptides
complexed with a 300-nucleotide (7S)molecule of RNA.
?ER signal sequence,Typically 15-30 amino acids:
Consist of three domains,a positively charged N-terminal region,a
central hydrophobic region,and a polar region adjoining the site where
cleavage from the mature protein will take place,A signal sequence on
nascent seretory proteins targets them to the ER and is then cleaved off
?SRP receptor (GTP binding protein)
SRP have three main active sites:
One that recognizes and binds to
ER signal sequence;
One that interacts with the
ribosome to block further
translation;
One that binds to the ER
membrane (docking protein))
The sorting signal
of VSV
glycoproteins,
Asp-X-Gln或 DXE
Figure 6-43,The sorting of proteins destined for the apical and basolateral plasma
membranes of epithelial cells,When cultured MDCK cells are infected simultaneously with
VSV and influenza virus,the VSV glycoprotein is found only on the basolateral membrane,
whereas the HA glycoprotein of the influenza virus is found only on the apical membrane,Like
these viral proteins,some cellular proteins are sorted directly to the apical membrane and others
to the basolateral membrane via specific transport vesicles that bud from the trans-Golgi network,
In certain other polarized cells,some apical and basolateral proteins are transported together to
the basolateral surface; the apical proteins then move selectively,by endocytosis and transcytosis,
to the apical membrane,[K,Simons et al.,Cell 62:207; K,Mostov et al.,JCB,116:577]
?Start-transfer Sequence &
Stop-transfer Sequence
Figure 6-24,Synthesis and insertion into the ER membrane of the GLUT1 glucose
transporter and other proteins with multiple transmembrane ?-helical segments.
The N-terminal ? helix functions as an internal,uncleaved signal-anchor sequence (red),directing binding of
the nascent polypeptide chain to the rER membrane and initiating cotranslational insertion,Both SRP and the
SRP receptor are involved in this step,Following synthesis of helix 2,which functions as a stop-transfer
membrane-anchor sequence,extrusion of the chain through the translocon into the ER lumen ceases,The first
two ? helices then move out of the translocon into the ER bilayer,anchoring the nascent chain as an ?-helical
hairpin,The C-terminus of the nascent chain continues to grow in the cytosol,Subsequent ?-helical hairpins
could insert similarly,although SRP and the SRP receptor are required only for insertion of the first
signalanchor sequence,Although only six transmembrane ? helices are depicted here,GLUT1 and proteins
of similar structure have twelve or more,[H,P,Wessels et al.,Cell 55:61.]
?The Orientation of Nascent Polypeptide
The Nascent polypeptide is oriented within translocon
so that the positively charged flanking sequence faces the
cytosol
C,A Model for the Postranslational Import
of Polypeptides into the Mit.
?Post-translational modification and quality control
in the rER
Disulfide bonds are formed and
rearranged in the ER lumen
Only in ER lumen is there a redox
environment for oxidation of –SH
groups.
PDI,protein disulfide
isomerase,found in abundance
in the ER lumen
?Correct
folding of newly
made proteins is
facilitated by
several ER
proteins
Proteins without
any signal
sequence are
cytosol residual
proteins
6,Types of Vesicle Transport and Their Functions
A,The three different types of coated vesicles.
Different coat proteins select different cargo and shape the
transport vesicles that mediate the various steps in the
biosynthetic-secretory and endocytic pathways.
?COPII-coated vesicles move materials from the ER
to the Golgi.
The assembly of a COPII-coated vesicles.
Sar—GTP binding protein,Sar-GTP binds to the ER; Sar-
GDP dissociates from the ER
Antibodies is
able to block the
budding of
vesicle from ER
but have no
effect on vesicle
transport from
one Golgi
compartment to
another in
mammalian cell.
? COPI-coated vasicles transporting Escaped ER
resident Proteins Back to the ER.
(1) The assembly of a COPI-coat is mediated by ADP-ribosylation
factor(ARF),GTP binding protein,which is required for vesicle
transfer between cisternae.
(2) COPI coated vesicles may select specific cargo.
?ER is an open prison,?Soluble ER protein bear Retrieving signal—
KDEL(Lys-Asp-Glu-Leu)in mammal and HDEL in yeast,whereas ER
membrane proteins bear the signal KKXX,?The KDEL receptor
present in vesicular tubular clusters and the Golgi apparatus..
(3) COPI-coated vesicle were first identified by treatment of GTP
analogues --- COPI-coated vesicle accumulated within the cell
and could be isolated by centrifugation.
A model for the retrieval of ER resident proteins.
The KDEL receptor captures the soluble ER resident proteins and
carries them in COPI-coated transport vesicles back to the ER.
Neutral pH,dissociate from the KDEL; low pH,binding the KDEL
? Clathrin-coated vesicle,Transporting Cargo from
the TGN to endosomes,Lysosomes,and plant
vacuoles and also move materials from the PM to
cytoplasmic compartments along the endocytic
pathway.
(1) The TGN of Golgi is the Sourse of Clathrin-coated vesicle.
(2) Clathrin-coats contain,
protein clathrin-----which forms a structural scaffold,
adaptors---- multisubunit,which forms an inner shell.
The formation of clathrin-coated
pits in the TGN
B,The SNARE Hypothesis for Transport
Vesicle Targeting and Fusion
?Specificity in vesicle
docking and fusion is thought
to be attained through
specific interactions between
specific v-SNARE proteins
on the vesicle membranes
and t-SNARE proteins on the
membranes of the target
compartment.
?SNAREs are a protein
family,There are at least 20
different SNAREs in an
animal cell.The role of SNAREs in guiding
vesicular transport.
The struction of paired SNAREs (the four-helix bundle).
The SNAREs responsible for docking synaptic vesicles at
the plasma membrane of nerve terminals consist of three
proteins.
The basic molecular components in eukaryotic cells include
v-SNAREs (v-SNAP receptors) on transport vesicles,t-SNAREs
(t-SNAP receptors) on target membranes,Rab GTPase,NSF,and
several SNAPs,SNAPs are soluble NSF attachment proteins.
The role of Rab proteins in
facilitating the docking of
transport vesicles.
GEF in donor membrane;
GTP binding alters the
conformation of Rab,exposing
its covalently attached lipid
group and helps anchoring;
Rab and Rab effectors help the
vesicle dock and pairing of the v-
and t-SNAREs;
GDI,GDP dissociation inhibitor.
kinesin dynein
C,Motor proteins mediate movement of
vesicles along MT
A General Model for Kinesin-and Dynein-
mediated Transport in a Typical Cell
D,Maintenance of membrane asymmetry
E,Membrane flow
作 业
? 膜泡表面标志与膜泡运输
? 过氧化物酶体中蛋白质的
来源及输入机制
