Vocab terms Flashcards
1
Q
Nucleus
A
largest organelle
1 per cell
responsible for:
1. compartmentalization of genome and its activities
ex. site of DNA replication, transcription and RNA processing
2. metabolism, protein synthesis, reproduction
2
Q
Nucleoid
A
The nucleoid (meaning nucleus-like) is an irregularly-shaped region within the cell of a prokaryote that contains all or most of the genetic material
3
Q
Nucleolus
A
most conspicious nuclear subdomain
where genes that encode rRNA are found, site of ribosome production
4
Q
Nucleoplasm
A
the cytoplasm of the nucleus
5
Q
transcription factories
A
where transcription factors are concentrated - located within interchromosomal channel
6
Q
interchromosomal channel
A
regions between chromosome subdomains that serve as barrier to prevent unwanted DNA - DNA and/or DNA protein interactions
7
Q
nuclear speckles
A
subdomains where mRNA splicing factors concentrated - in interchromosomal channels next to transcription factories
8
Q
nuclear matrix
A
insoluble fibrillar-like protein network (mesh) distributed throughout nucleoplasm - provides shape of nucleus = cytoskeleton network of nucleus
3 parts: microfilaments, microtubules and intermediate things
9
Q
nuclear envelope
A
separates contents of nucleus from cytoplasm, serves as barrier, composed of:
nuclear membrane, lamina and pore complexes
10
Q
inner nuclear membrane
A
unique protein composition just the inner membrane bruh
11
Q
outer membrane
A
continuous w RER - ribosomes attached to cytoplasmic surface of outer membrane
12
Q
nuclear envelope lumen
A
continuous w ER lumen - the space btw the outer and inner membranes
13
Q
nuclear lamina
A
network of mesh filaments called lamina located on inner surface of nuclear inner membrane
- mutations in lamin result in progeria
- provides mechanical support to nuclear envelopes and serves as scaffold for attachment of chromatin and nuclear matrix to nuclear envelope
14
Q
ABC nuclear lamins
A
proteins that form intermediate filaments in cytoskeleton network - proteins that make up the nuclear lamina
15
Q
NPC
A
nuclear pore complex
channels/doorways in nuclear envelope formed where outer and inner membrane join, 3000-4000 per nucleus, composed of 40 nups
16
Q
Nups
A
nucleoporin
integral and peripheral inner and outer nuclear membrane proteins
17
Q
central scaffold
A
composed of integral / trans membrane bound nups - anchors NPC to nuclear envelope membranes - forms aqueous central channel (like tube)
18
Q
central channel
A
aqueous channel between central scaffold of nuclear pore
19
Q
Y complex
A
cytoplasmic and nuclear side of NPC, respectively
- linked to central scaffold and cytoplasmic filaments or nuclear basket
20
Q
cytoplasmic filaments
A
long, filament shaped nups that extend into cytoplasm - involved in receptor - cargo recognition
21
Q
nuclear basket
A
basket like structure located on nuclear side of NPC - linked to Y complex nuclear ring
- involved in nuclear receptor cargo - protein import
22
Q
FG nup
A
FG domains that extend into central channel
form mesh that limits diffusion of macromolecules greater than 40 kDa
mlc greater than 40kDa are unable to pass through NPC freely
FG nups have an unusual amino acid sequence, highly disordered 2* structure, extended, flexible organization fills central channel
the curvy part is hydrophilic region
the dots are FG repeats
23
Q
NLS
A
nuclear localization signal
specific stretch of amino acids recognized by nuclear receptor proteins
allows translocation of protein w NLS into nucleus from cytoplasm
24
Q
Classic NLS
A
most common - short stretch of positively charged basic amino acid residues
25
Bipartile NLS
2 short stretches of basic aa and 7-10 aa-long 'spacer' sequence
- proteins can have more than one NLS aa sequence that is both necessary and sufficient
26
karyoferins
transport things into (importin) or out of (exportin) the nucleus
large family of receptor proteins
27
importin
karyoferin, consists of importin a and b
- brings things into the nucleous
28
importin alpha
subunit of importin that recognizes and binds to basic residues in cargo proteins NLS
dissociates in nucleus when Ran-GTP binds importin b
29
importin beta
binds to cytoplasmic filaments at NPC and Ran GTP
subunit of importin
30
Ran-GTP
active GTP bound form of Ran
higher concentration in nucleus
(maintained thanks to GEF)
31
Ran-GDP
inactive GDP bound form
high concentration in cytoplasm
maintained by GAP
32
NES
nuclear export signal - specific stretch of amino acids recognized by exportin - necessary and sufficient
leucine-rich NES - most common NES consists of leucine-rich motif LxxLxxL
x = any amino acid residue
33
piggyback nuclear import
some proteins don't have NLS so they ride proteins with one to get into nucleus
34
cyclin
nucleoplasmic proteins involved in control of cell cycle
35
cell cycle
interphase (G, S, and G2) and M phase (mitosis and cytokinesis)
36
CDKs
cell-cycle specific kinase enzymes
located in nucleus; phosphorylate various, target (nuclear) proteins --> turn 'on' or 'off'
37
open mitosis
nucleus completely disassembled by metaphase
38
proteosome
protein degradation machinery
39
brightfield microscopy
light diffracted by specimen and undiffracted light focused by objective lens
image captured by video camera, easily manipulate digital images using computer software programs
poor contrast, less detail, specimen's fixed
40
deconvolution
designed to remove background and out of focus light (yields increased contrast and clarity)
41
magnification
purpose of microscopy --> to generate magnified, high quality view of specimen
overall magnification = objective lens x ocular lens
42
resolution
minimum distance that can seperate two points that still remain identifiable as seperate points
- most important aspect of today's microscopy
resolving power depends on:
1. wavelength of illumination light
2. numerical aperture
43
NA
numerical aperture
light gathering qualities of objective lens and specimen mounting medium
44
resolution formula
0.61 x lambda / NA
45
Fixation
formaldehyde fixation - cross links amino groups on adjacent proteins/nucleic acids, embedded for support, then sectioned with microtome and strained with molecule-specific dyes
RESULTS in DEATH
46
microscopy artifacts
fixation results in cell death embedding and sectioning can lead to structural artifacts
47
microtome
mini knife machine thing that slices specimen into cross sections
48
fluorescence microscopy
microscopy technique for visualizing molecules in living (or fixed specimens)
- relies on autoflurexcence or immunofluoreschence
49
autofluorescence
endogenous fluorescence in specimen
50
immunofluorescence
applied fluorescent dyes or dye - conjugated antibodies
51
autofluorescent proteins
another fluorescent method
52
CLSM
confocal laser scanning microscopy
method of fluorescence microscopy - similar set up as brightfield but with additional features
one or more laser of certain wavelengths of light excite fluoresant molecules in specimen and emitted light specifically focused to obtain detailed image
specimen rapidly scanned with point laser light
emitted flueorescent light from single layer (focal plane)
yields 2D s-section (optical slice) / focal plane
53
z - stack
stack of individual z-sections collected at different depths in sample and combined to generate 3D image
54
photobleach
limitation of CLSM - point laser can remove fluorescence of sample / photo bleach
55
phototoxicity
damage to live cells due to point laser from CLSM
56
Super resolution CLSM
10 X better resolution than CLSM - useful for visualizing smaller intra-cellular structures
57
transport vesicles
membrane bound vesicles that transport large amounts of meterial btw each organelle/structure in endomembrane system
58
donor membrane
where vesicle buds off from / sender
59
acceptror membrane
receptor/target of vesicle - where vesicle contents are delivered (vesicle fuses with it)
60
Biosynthetic pathway
materials (like lysosomal proteins) transported from ER to Golgi, endosomes and then lysosomes (vacuoles in plants)
- or from endosomes to plasma membrane and extracellular space
61
Constitutive secretion
materials continually transported from Golge it pm and/or released via exocytoses out side of cell in a secretory VESICLE
62
secretory vesicle
type of vesicle, smaller, used in constitutive secretion
63
exocytosis
vesicle trafficking to and fusion with pm and release of contents
64
regulated secretion
occurs only in specialized cells
ER derived materials from Golgi are stored in secretory granules and released in a regulated manner
65
secretory granule
used in regulated secretion
66
endocytic pathway
operates in opposite direction of secretory pathway
materials from plasma membrane and/or extra - cellular space incorporated into cell and then transported to endosomes and lysosomes
67
endocytosis
uptake of materials from pm and ectracellular space into transport vesicles
68
pulse chase labelling and autoradiography
pulse - tissue briefly incubated with radioactive amino acids which are incorporated into newly-synthesized proteins
chase-tissue washed and incubated for varying lengths of time with non-radioactive amino acids
69
autoradiography
tissue fixed (killed) and exposed to X-ray film
where the radioactive protein is located determines pathway
results: brief chase = ER
intermediate chase = Golgi
long chase = secretory vesicles
70
Live-cell imaging using autoflueorescent proteins
GFP linked to gene of interest and gene introduced to cell via recombinant gene fusion - can also use temperature sensitive viral glycoprotein fused to GFP and introduce it into cell to visualize movement of proteins
71
subcellular fractionations
uses centrifugation - separate and purify specific organelles on the basis of their varying sizes and/or densities
72
homogenization
cell/tissue disrupted by gentle homogenization (ensures organelles remain intact)
a cell's structure is broken down to release its internal components uniformly into its surrounding medium
73
homogenate
the filtrate after initial centrifugation that's subjected to differential centrefugation
74
supernatant
liquid at top of centrifuge tube that's subjected to further centrifugation
75
differential centrifugation
seperates intact organelles/cellular components of different size/density with increasing higher speeds of centrifugation
76
microsomes
fragments of ER membrane (and / or plasma membrane) that fuse and reform into small, spherical vesicles
individual organelles in each pellet fraction (mizture of organelles) can be further purified
77
equilibrium density
gradient centrifugation - separates intact organelles / cellular components on basis of density
78
sucrose gradient
used in equilibrium density-gradient centrifugation - differing concentrations of sucrose that organelle fraction layered on top of
79
cell-free (in vitro assays)
characterization of the activities (funcitons) of specific endomembrane protein components in vitro - allows for study of cellular processes without cell
80
liposomes
artificial, spherical vesicles consisting of phospholipid bilayer surrounding aqueous center that isolated proteins are incubated with
liposomes mized with purified proteins to see their effect
ex. proteins involved in formation of transport vesicles caused budding
81
mutant phenotype analysis
approach of studying / identifying genes and proteins involved in vesicle trafficking by screening for mutant phenotypes
ex. yeast 'sec' mutants - collection of ts mutants that secrete proteins at permissive temps but not at higher (restrictive) temps
accumulate normally secreted endomembrane proteins at points in endomembrane path that are blocked by mutation
Class A --> defect in transport to ER
Class B --> defect in budding of vesicles from rough ER, accumulation in rough ER
82
Endoplasmic reticulum
starting point for both secretory and biosynthetic pathways - site of protein and lipid synthesis, protein folding and processing / quality control
highly complex network of membrane - enclosed, rod-like tubules and sheet-like cisternae
organelle with largest surface area
83
ER tubules
ER rod like structures of ER like spaghetti
84
ER cisternae
flattened sacs of ER that increase SA
85
ER lumen
aqueous space inside ER
86
Reticulons
major protein/integral membrane protein in ER that is responsible for maintaining shape of tubules and cisternae
possess unique hair-pin (v-shaped) 2* structure = regulate ER membrane curvature and overall shape of ER
87
ER subdomains
distinct regions of ER that possess unique functions and/or morphologies
88
RER
rough ER - a subdomain - mostly cisternae w bound ribosomes, involved in protein and membrane phospholipid synthesis
89
SER
mostly curved tubules lacking ribosomes, involved in Ca2+ storage, and hormone synthesis
90
nuclear envelope
outer nuclear membrane continuous with RER, contains nups and attatched ribosomes
91
MAM
mitochondria associated membrane
a subdomain of the ER that makes direct contact with mitochondria and is involved in membrane protein exchange and fission!
92
PAM
Subdomain of ER
plasma membrane associated membrane - makes direct contact w pm, involved in membrane protein and lipid exchange
93
ERES
ER exit sites - ER regions where transport vesicles bud off enroute to Golgi
94
free ribosomes
ribosomes in cytoplasm where protein translation begins
95
membrane-bound ribosomes
ribosomoes in ER membrane where soluble or membrane protein in ER made
96
Co-translational translocation
protein targetting to and across ER membrane
97
SRP
signal sequence that soluble ER proteins and Type I ER membrane proteins have
it's recognized by the ER receptor
98
SRP receptor
hetero-dimeric ER integral membrane protein complex
cytoplasmic face is docking site for incoming SRP
interaction between SRP and SRP receptor stregthened by both binding GTP, GTP hydrolysis, results in release of SRP
99
Sec61
a multiprotein translocon complex that forms an hourglass shaped aqueous channel/ translocon channel
100
Sec61 pore ring
ring of 6 hydrophobic amino acids located at narrowest diameter of channel - serve as gate to seal channel (pore ring)
its also blocked by an alpha helix plug
101
alpha helix plug
Sec61 translocon channel is also blocked by an alpha helix plug that moves out of the way during protein translocation into ER
102
signal peptidase
N-terminal signal sequence enters ER lumen and is cleaved by this integral membrane protease associated w Sec61 translocon
it recognizes cleavage sequence motif at c-terminal end of signal sequence
103
Reticuloplasmins
ER molecular chaperones that mediate glycosylation (oligomeric assembly) and proper protein folding
include BiP, calnexin, and calreticulin
104
membrane protein topology
number of membrane spanning domains and orientation
like it describes type I to IV proteins
105
TMD
transmembrane domain
typically alpha helic of 16-25 hydrophobic amino acids (energetically favourable within hydrophobic interior of phospholipid bilayer)
106
Type I integral membrane protein
has signal sequence that's cleaved (only integral membrane protein with signal sequence N-terminal)
has STA and is oriented
N in - C out
107
Type II integral membrane protein
Has SA sequence and +ve aa near N-terminal
it's oriented Cin - N out
108
Type III integral membrane
has SA sequence, +ve aa located downstream (C-terminal) of SA
oriented Nin - Cout
109
Type IV
multispanning integral membrane protein
multiple TMDs - contains both SA sequence and STA sequences
110
STA
stop transfer anchor sequence - TMD that stops further translocation of polypeptide through translocon
111
ER export sorting signal
di-acidic (-Asp-x-Glu-) ER export signal is the most comon
- how selection (recognition) of vesicle membrane proteins by Sec24 is mediated
located on cytoplasmic facing domain of Sec24-selected vesicle membrane proteins
ER export sorting signals are not found on ER resident proteins
112
Sec 13 and Sec 31
self-assemble into outer, cage-like lattice and act as structural outer scaffolding for growing COPII vesicle bud
- recruited by Sec23/24
- promotes additional outward bending of ERES membrane
113
cis-Golgi network
CGN
vesicles trafic from ERES to here
incoming nascent vesicles fuse with one another to form CGN
consists of interconnected network of vesicles and tubules located on cis face (side) of Golgi complex
114
Rab proteins
large family of lipid membrane-anchored, GTP-binding proteins associated with all transport vesicles
key regulators of vesicle trafficking and fusion
activated Rab (Rab-GTP_ binds to specific Rab effector(s) proteins on target membrane
115
Rab effectors
Proteins on target membrane that Rab binds to to dock vesicles
form molecular bridge (rab bound to rab effector)
116
Molecular bridge
unique vesicle Rabs associate with Rab effectors on specific target membranes
117
SNARE proteins
large family of integral membrane-bound proteins located on all transport vesicles AND all target membranes
include v-SNAREs and t-SNAREs
118
v-SNAREs
found on transport vesicle (v) membranes
incorporated into vesicle membrane at site of budding on donor membrane compartment
119
t-SNARES
found on target (t) ‘acceptor’ membranes
120
SNARE motif
all SNARE proteins possess SNARE motif
cytoplasmic-facing, coiled-coil domain in both v- & t-SNAREs that extend from vesicle/target membrane surface
121
SNARE complex
what its called when v and t SNAREs coil together
SNARE motifs in cognate v-SNARE and t-SNAREs interact to form stable SNARE complex
pulls vesicle and target membranes close together
122
SA sequence
signal anchor sequence
functions as signal sequence for binding SRP and mediating nascent polypeptide-ribosome complex targeting to translocon and membrane anchor
123
positive-outside rule
the positive amino acid residues are always outside (cytosol) and this determines orientation of most membrane proteins synthesized at RER
124
membrane bilayer asymmetry
nascent ER membrane proteins and lipids distributed and/or oriented in assymetrical manner
integral membrane proteins - diff. regions of protein located on either cytoplasmic or exoplasmic
125
N-liked glycosylation
addition of specific short chains of sugar monomers (liked together in a specific order to form oligosaccharide) to terminal amino group of asparagine (N)
includes:
i) core glycosylation
ii) core modification
126
core glycosylation
glycosyltransferases (in ER) synthesize core oligosaccharide
complex process - hihgly branched oligosaccharide chain consisting of 14 sugar residues, including mannoses and 3-glucore-long terminal branch
begins with addition of first sugar to dolichol phosphate
glycosyltransferases continue to add sugars at specific positions on growing core oligosaccharide
final step --> transfer of core oligosaccharide from dolichol lipid carrier to nascent soluble/ membrane protein while being synthesized
core oligosaccharide transferred to lumenal facing portions of nascent ER proteins with specific amino acid sequence motif -N-x-S/T-
127
Core modification
second stage of N-linked glycosylation
attached 14 sugar (core-oligosaccharide) is trimmed and modified
2/3 terminal glucose units removed by glucosidase
subsequent removal (and re-addition) of last glucose unit important for proper protein folding/assembly
during N-linked glycosylation and modification, nascent protein rapidly folded into proper 3D conformation - mediated by reticuloplasmins and PDI
128
glucosidases
2/3 terminal glucose units removed by glucosidases
ER lumenal protein that removes the last glucose unit too
this is during core modification
129
PDI
protein disulfide isomerase
catalyzes formation / intracellular disulfide bonds
disulfide bonds btw cysteine residues on same or different nascent polypeptides promote proper folding and assembly by stabilizing their proper 3D conformation
130
ER protein quality control
reticuloplasmins and PDI bind to nascent glycoprotein while its being synthesized and help mediate proper folding, oligomeric assembly, stability, etc.
if protein is properly folded - one mannose is removed by ER mannosidase
if protein is misfolded - its recognized by UGGT monitoring enzyme - which adds back single glucose unit to oligosaccharide core
process continues till ptorein is properly folded
131
mannosidase
removes one mannose unit during ER protein quality control if its properly folded - in ER lumen
132
UGGT
a monitoring enzyme
glucotransferase
serves as a protein conformation sensing protein - recognizes hydrophobic residues of misfolded protein and adds back single glucose unit
133
ERAD pathway
uses AAA ATPase p97 - ER membrane protein
uses ATP hydrolysis to pull misfolded protein accross ER membrane into cytosol - retrotranslocation
134
AAA ATPase p97
ER membrane protein
uses ATP hydrolysis to pull misfolded protein accross ER membrane into cytosol - retrotranslocation
135
UB - ubiquitin
small (76 aa) protein involved in diverse cellular functions = mono and poly UB
136
mono - UB
signal for targeting membrane proteins into intralumenal vesicles of late endosomes/ multivesicular bodies
137
poly-UB
serves as signal for ER protein degradation and for most other cellular proteins destined for normal turnover
138
proteosome
degrade poly UB proteins
barrel shaped, multi-subunit machine
139
proteosome degredation process:
1) UB protein binds to cap or lid of proteosome
2) Poly-UB chain removed (recycled)
3) protein threaded into proteasome and degraded (via proteolysis)
4) free aa s are reused for new protein synthesis
140
proteiolysis
the process by which protein is degraded in the proteosome
141
UPR
unfolded protein response pathways
if there's an overloaded ERAD - ER stress signal
each pathway mediated by unique protein sensor - ER integral membrane-spanning proteins
IRE1, PERK, ATF6
142
PERK
an unfolded protein response pathway
BiP is released form PERK in stress (to aid in folding of accumulated ER proteins)
PERK dimerizes to become active
cytoplasmic-facing kinase domain of activated PERK dimer phosphorylate (inhibit) eIF2a
this causes a decrease in translation and overall protein synthesis
143
eIF2a
protein translation factor required for initiation of protein synthesis
participates in ribosome mRNA binding
144
ATF6
in ER-stress conditions, BiP released from ATF6
active ATF6 moves from ER to Golgi
at Golgi, cytoplasmic-facing Tf domain is cleaved by a Golgi associated protease
in nucleus, ATF6 tf domain upregulates genes encoding key proteins involved in ER quality control
145
Sar1
a soluble COPII component (GTPase) recruited from cytoplasm to ER membrane via binding Sec12
it binds GTP causing conformational change - exposing anchor
it also binds/recruits several other COPII proteins from cytosol to ERES membrane surface
146
Sec23/Sec24
bind Sar 1 and soluble COPII proteins form ternary complex with Sar 1 at ERES
they act as structural scaffolding and promote initial outward bending of ERES membrane
147
Golgi complex
unique morphology –‘complex’ or ‘stack’ of flattened, membrane-bound cisternae (sacs) with dilated edges and numerous associated
tubules and vesicles
possesses several subcompartments:
cis, medial and trans
CGN and TGN
148
CGN
located at cis face of Golgi complex
* consists of complex, interconnected network of tubules
and vesicles adjacent to ERES
* initial destination of COPII transport vesicles from
ERES
serves as a ‘sorting station
149
what sites of CGN are there?
destination (‘acceptor’ compartment) of COPII vesicles coming
‘forward’ (anterograde transport) from ERES to CGN
and
site of COPI vesicle assembly for transport ’back’ (retrograde
transport) from CGN to ER
and
‘forward’ (anterograde) transport as CGN matures into next
subcompartment of Golgi complex (i.e., CGN → cis cisternae)
and
destination of COPI vesicles moving ‘back’ (retrograde transport)
from next subcompartment of Golgi complex (cis cisternae)
to CGN
150
Golgi cisternae
series of three or more large, flattened cisternae
they comprise the majority of the golgi
includes cis, medial and trans
sites of Golgi metabolism
151
trans-Golgi network (TGN)
located on trans face of Golgi complex
* interconnected network of tubules and vesicles ( CGN)
* serves as ‘sorting station’ ( CGN)
152
what are the sites of TGN for
‘forward’ (anterograde) transport as previous subcompartment
of Golgi complex matures into TGN (i.e., trans cisternae → TGN)
and
site of clathrin coat vesicle assembly for transport ‘forward’
(anterograde transport) from TGN to endosomes
153
Golgi matrix
various Golgi peripheral and integral membrane proteins
cytoplasmic-facing domains interact to form 'scaffold' - link CSN, cis/medial/trans cisternae, and TGN tg
organization of Golgi complex mediated by Golgi matrix
links Golgi complex to cytoskeleton
154
GRASPs
Golgi reassembly and stacking proteins
part of Golgi matrix
serve as tethering proteins
link different Golgi subcompartments together
155
glycosylation in Golg
N-linked glycosylation completed in Golgi complex
* cis, medial and trans Golgi cisternae possess unique glycosyltransferase and glycosidase enzymes
additional modification of glycoprotein’s N-linked core
oligosaccharide(s) required for proper protein function and/or
targeting
* Golgi cisternae act as ‘assembly line’
156
a-mannosidase I
is an enxyme in cis cisternae that
removes 3 mannose sugars from core oligosaccharide of glycoprotein
157
M6P
mannose 6 phosphate
mannose-6-phosphate (M6P) group(s) serve as
soluble lysosomal protein targeting signal
mannose units of a protein destined for lysosome are phosphorylated in cis cisternae of Golgi
in cis cisternae, mannose units in core oligosaccharide(s) of soluble
proteins destined for lysosomes are phosphorylated
they are phosphorylated by N-acetylglucosamine phosphotransferase
proteins w M6P are packaged at TGN into clathrin-coated transport vesicles to endosomes and then lysosomes
lysosomal targeting signal
proteins with M6P: TGN → clathrin-coated vesicle → late endosome → lysosome
158
N-acetylglucosamine phosphotransferase
recognizes unique sequences
in lysosomal-destined proteins – “signal patch”
adds phosphate to mannose to make M6P
159
Cisternal progression/
maturation model
Golgi subcompartments are dynamic structures
each subcompartment continually moves (forward) from
cis to trans side of Golgi complex
subcompartment movement mediated by Golgi matrix
proteins and cytoskeleton (motors)
* composition of each subcompartment changes while moving cis to trans through complex
e.g., CGN matures into cis cisternae, cis cisternae matures into medial cisternae, etc
* overall, Golgi complex persists (structurally & functionally) because COPI transport vesicles continually move resident Golgi proteins ‘back’ (retrograde transport)
to proper subcompartment
160
COPI
is a vesicle coat that coats vesicles moving in retrograde direction from trans to cis Golgi
overall, Golgi complex persists (structurally & functionally)
because COPI transport vesicles continually move
resident Golgi proteins ‘back’
like bringing a-mannosidase I back to cis cisternae
161
What are the 4 types of transport vesicles that TGN disperses into
clathrin-coated vesicles – with M6P-bearing protein ’cargo’ target
to endosomes (lysosomes)
secretory vesicles – target to plasma membrane (extracellular space)
secretory granules – eventually target to pm (extracellular space)
COPI-coated vesicles – target (‘back’) to trans Golgi cisternae
162
Lysosome
‘digestive’ organelle – degrades all types of macromolecules (e.g., lipids, sugars, nucleic acids & proteins
also plays key role in degradation of larger cellular components/ organelles – autophagy
contains ~60 different soluble acid hydrolyase enzymes
low pH in lysosomal lumen maintained by membrane-bound ATPase proton pumps
highly dynamic – lysosomes possess wide variety of shapes and sizes depending on organism/tissue/cell type
163
autophagy
role of lysosome
= degradation of larger cellular components/ organelles
164
acid hydrolyase
enzymes in the lysosome (there are aprrox. 60)
enzymatically active only at low pH (4.6) of lysosome interior lumen
165
M6P receptor
integral transmembrane protein in TGN membrane
recognizes M6P-bearing lysosomal destined proteins
integral transmembrane protein
lumenal-facing domain of M6P receptor binds
to M6P groups on soluble lysosomal-destined
proteins in lumen of TGN
* M6P receptor mediates subsequent
concentration of soluble lysosomal (‘cargo’)
proteins into nascent clathrin-coated
transport vesicles
cytoplasmic-facing domain of M6P
receptor binds to AP1 and GGA adaptor
coat proteins
166
AP complex
AP1 and GGA
adaptor coat proteins
mediate vesicle cargo selection
serve as linker for clathrin-coat vesicle assemble at TGN membrane
bind Arf1, cytoplasmic-facing domain of MP6 and clathrin!
recruited by Arf 1
167
Arf 1
recruits AP1/GGA adaptor proteins from cytoplasm to TGN surface
Arf1 also involved in initiation of COPI-vesicle assembly during retrograde transport from and within Golgi complex
* Arf1 binding to GTP causes conformational
change
exposed lipid anchor in Arf1-GTP directs it from
cytoplasm to outer leaflet of TGN membrane;
initiates outward bending of membrane
168
Clathrin
ne molecule of clathrin consists of
three ‘light’ chain polypeptides & three
‘heavy’ chain polypeptides
* form three-legged structure: triskelion
* clathrin triskelions recruited from
cytoplasm self-assemble to form outer
‘scaffolding’ (cage-like lattice) of ‘coat’ on
growing vesicle
inner layer of the coat consists of AP complex
linked to Arf1 and M6P receptor bound to
soluble lysosomal-destined ‘cargo’ proteins
clathrin ≈ Sec13/31 outer scaffolding in
COPII vesicle formation at ERES
clathrin assembly promotes curvature (outward bending) of TGN membrane
* individual clathrin triskelions initially self-assemble to form hexagons that lie flat
on membrane (cytoplasmic) surface
* triskelions subsequently self-assemble to form pentagons
transition to hexagons and pentagons serves as mechanical driving
force for membrane curvature
169
dynamin
large, soluble GTP-binding protein
dynamin recruited from cytosol to connection (stalk) btw growing clathrin-coated byd and TGN membrane
dynamin proteins self-assemble to form dynamin ring around stalk
GTP hydrolysis causes conformational change in dynamin ring resulting in twisting and pinching off/ scission
170
scission
when a vessicle is released due to 'pinching off' like by dynamin or dynamin related proteins
171
What happens to vesicle after pinching off from TGN
- clathrin coat disassembles
- Arf1-GTP is converted to Art1-GDP and that conformation change
AP complex and clathrin triskelions are released into cytoplasm and rexycled for additional rounds of clathrin-coat assembly at TGN
172
gamma GTP
non-hydrolyzable analog of
GTP) causes continued dynamin ring polymerization –
results in long, extended ‘stalk’ and no scission of vesicle bud
so basically, Arf-GTP gamma cannot hydrolyze to Arf-GDP and so the dynamin ring just keeps growing and growing off the TGN membrane and the stalk hets longer and longer (that weird spiral picture)
173
Late endosome
nascent (uncoated) vesicle with M6P-receptor
bound soluble lysosomal ‘cargo’ proteins
fuses coming from the TGN or the pm fuses with late endosome
at late endosome...
* acidic interior (~pH 5.0-5.5 in lumen) of
late endosomes causes M6P receptors to dissociate from soluble lysosomal ‘cargo’ proteins (acid hydrolases)
TGN & TGN-derived vesicles = ~pH 6.5
* phosphate removed from M6P groups in
soluble ‘cargo’ proteins = prevents rebinding to M6P receptor
empty’ M6P receptors ‘recycled’ back
to TGN via retromer-coated vesicles
and retromer complex coat assembles on cytoplasmic surface of late endosome
Late endosomes: junction of biosynthetic and endocytic pathways
* materials derived from plasma membrane and extracellular space via receptor-
mediated endocytosis (endocytic pathway) delivered to endosome/lysosome for
internalization/degradation
late endosome eventually becomes multivesicular body and fuses with lysosome
174
Retromer transport vesicle
empty’ M6P receptors ‘recycled’ back
to TGN via retromer-coated vesicles
[Step 4]
recently-discovered protein ‘coat’ (≈ COPI/II & clathrin)
* retromer complex ‘coat’ assembles on
cytoplasmic surface of late endosome
mediates membrane curvature and vesicle
budding (how?) AND selects proper vesicle
‘cargo’
* retromer ‘coat’ disassembles after
vesicle formation at late endosome
retromer vesicles (w/ various pm cargo and
‘empty’ M6P receptors) also traffic to
plasma membrane
175
retromer complex
retromer complex ‘coat’ assembles on
cytoplasmic surface of late endosome
mediates membrane curvature and vesicle
budding (how?) AND selects proper vesicle
‘cargo’
the coat disassembles after vesicle formation at late endosome
176
receptor-
mediated endocytosis
= endocytic pathway
materials delivered from plasma membrane and extracellular space via receptor-mediated endocytosis are delivered to endosome/lysosome for internalization/degredation
177
Constitutive secretion pathway
Considered default pathway
materials continually transported (via secretory vesicles) from TGN to pm
vesicles fuse (via Rabs & SNAREs) with pm and release (exocytosis) their lumenal soluble ‘cargo’ outside of cell
vesicle mebrane 'cargo' components are incorporated into plasma membrane
178
Regualted secretion pathwaay
materials at TGN packaged into
secretory granules
* in response to cellular signal...
granules stored in cytoplasm target to and fuse with (via Rabs
and SNAREs) plasma membrane and
release (exocytosis) their lumenal
‘cargo’ outside of cell
e.g., release of hormones by endocrine cells, neurotransmitters by nerve cells
179
Phagocytosis
one of the two main processes for internalization by the endocytic pathway
uptake of large, particulate materials from extracellular space by specialized cells
involves recognition via Fc domains on antibodies, Fc receptors on cell and the reassembly of actin microfilament network to engulf the foreign particle
180
Fc receptor
on the plasma membrane of a leukocyte - recognizes Fc domains on antibodies to help mediate phagocytosis
181
pseudopod
alterations in cytoskeleton result in changes in shape of
of leukocyte – cell extensions = pseudopods
happens during phagocytosis
Fc receptors ‘signal’ re-assembly of actin microfilament network
182
Fc domain
is a domain on an antibody that is recognized by Fc receptors on the cell about to perform phagocytosis
183
Phagosome
leukocyte pm (pseudopods) engulf bacterium [Step 3] and fuse to form phagosome – internalized pm =
phagosome membrane
184
Bulk phase endocytosis
the second type of internalization in the endocytic pathway
= pinocytosis or cellular drinking
non-specific uptake of extracellular fluids and plasma
membrane proteins and lipids into small vesicles
pm recycles every ~20-90 min
185
receptor mediated endocytosis
Receptor-mediated endocytosis
specific cell-surface (pm) receptor binds extracellular
ligand(s) and receptor-ligand complexes subsequently
concentrated and internalized in clathrin-coated transport vesicles
transmembrane receptor at plasma membrane ‘activated’ by binding to specific
extracellular ligand
ytoplasmic-facing domain of receptor binds to AP2 adaptor ‘coat’ protein – cytoplasmic
protein serves as ‘linker’ during clathrin-coat vesicle assembly at pm
receptor-ligand-AP2 complex accumulates in clathrin-coated pit
nner (cytoplasmic) leaflet of pm at coated pit enriched in unique membrane phospholipids
membrane lipid ‘microdomain’
AP2 also recruits clathrin triskelions from cytoplasm
clathrin-coated vesicle bud ‘pinches off’ from pm via dynamin (and GTP hydrolysis) [Step 2]
* soon after budding, the clathrin coat disassembles from vesicle
nascent, uncoated endocytic vesicle referred to as early endosome
186
AP2
just like / analogous to AP complex but its for receptor mediated endocytosis - so happens at plasma membrane
P2 has multiple binding domains: i) PI(4,5)P2, ii) cytoplasmic-domains of pm transmembrane
receptors (w/ extracellular-bound ‘cargo’), and iii) clathrin
* similar to AP1/GGA adaptors at TGN, AP2 at cytoplasmic face of coated pit forms
inner layer of ‘coat’
* AP2 also recruits clathrin triskelions from cytoplasm
helps form/shape the early endosome
187
clathrin coated pit
receptor-ligand-AP2 complex accumulates in clathrin-coated pit
specialized regions (indentations) of pm where receptor-ligand complexes are concentrated
and endocytic vesicles eventually form
188
membrane lipid ‘microdomain'
inner (cytoplasmic) leaflet of pm at coated pit enriched in unique membrane phospholipids
membrane lipid ‘microdomain’ – enriched in phosphatidylinositol (PI) (4,5) P2 – serves as signal for recruiting AP2 with bound receptor-ligand into coated pit
189
phosphatidylinositol (PI) (4,5)
for
ms membrane lipid mimcrodomain for receptor mediated endocytosis
enriched in phosphatidylinositol (PI) (4,5) P2 – serves as signal for recruiting AP2 with bound receptor-ligand into coated pit
190
MVB
a late endosome with inward budding vessicles
degradation of endocytosed membrane proteins (e.g., cell-surface receptors) involves inward budding of vesicles into late endosome interior forming an MVB
MVB has unique morphology - contains numerous intralumenal vesicles
similar in size to transport vesicles, but opposite topology: MVB vesicles bud away from the
cytoplasm (unlike COPI/II, clathrin & retromer vesicles - bud towards cytoplasm)
* MVB vesicles selectively contain membrane proteins destined for degradation in
lysosome interior
membrane ‘cargo’ protein selection and inward (vesicle) budding mediated by ESCRT machinery
191
ESCRT
multi-protein complex – soluble (cytosolic) protein constituents recruited to MVB surface - mediate
membrane ‘cargo’ protein selection and inward vesicle budding – 5th class of ‘coat’ protein
mediate inward vesicle budding of late endosome / multivesicular bocies
Hrs recruits the ESCRT proteins
192
Hrs
recruits ESCRT machinery
signal for recognition by ESCRT protein Hrs
Hrs also mono-ubiquitinated
Hrs assemble at inward budding site
193
Vps4 ATPase
Disassembles the ESCRT complex (after the inward vesicles were formed in multivesicular body)
ESCRT components released into cytoplasm and recycled for additional rounds of MVB formation
194
HIV Gag protein
functions similar to Hrs
major structural viral particle protein at pm is mono-ubiqutinated and, like Hrs at MVBs, subsequently binds to and recruits (‘hijacks’) ESCRT machinery to plasma membrane
195
Mitochondria
The powerhouse of the c3ll LOLLLL
- semautonomous
- arise only from pre-existing organelles
serve essential & unique roles in eukaryotic cells....generation of metabolic energy (ATP)
energy production derived from carbohydrate & lipid catabolism (via oxidative phosphorylation [TCA cycle])
also unique from all other organelles....contain their own DNA (genome)
double membrane bound organelle
196
Chloroplasts
energy production and carbohydrate synthesis via photosynthesis
serve essential & unique roles in eukaryotic cells....generation of metabolic energy (ATP)
sort of like the mitochondria for plant cells
semi-autonomous
- arise only from pre-existing organelles
197
organelle biogenesis
includes protein targeting, membrane assembly, morphology, motility,
replication, degradation, and inheritance during cell division
198
semi-autonomous
describes mitochondria and chloroplasts
organelle replication controlled by both nuclear genome and organelle genome/ their own DNA SO COOOOLIO
199
Mitochondria outer membrane
permeable to ions & small molecules
contains porins – ‘barrel-shaped’ integral membrane proteins with large internal channe;
200
porins
‘barrel-shaped’ integral membrane proteins
with large internal channel
on outer mitochondrial membrane and allow passage of things inside
201
intermembrane space (mitochondria)
space btw outer and inner membranes
has a high H+ concentration
202
inner mitochondrial membrane
lies adjacent to outer membrane - forms ‘folds’ (cristae) that extend into organelle’s interior (provide increased surface area)
impermeable – maintains H+ gradient, site of ATP synthase
203
how do outer and inner mitochondrial membranes differ?
outer membrane - permeable
inner membrane - impermeable
outer and inner mitochondrial membranes differ functionally and overall protein & lipid composition
204
mitochondrial matrix
aqueous interior of mitochondria
within inner membrane
site of TCA cycle, ATP from oxidative phosphorylation
contains mitochondrial genome – circular DNA, varies between species in size, copy and gene number
human: encodes 13 proteins, 2 rRNAs & 22 tRNAs
contains ribosomes – used for translation of
mitochondrial genome-encoded proteins
205
mitochondrial network
highly branched, long & interconnected
series of tubules
highly dynamic structure
mitochondrial tubules are mobile and undergo their own fusion and fission in response to environmental stimuli, developmental status, and/or overall energy requirements of cell
mitochondrial network’ allows for cell-wide co-ordination of organelle’s functioning
and biogenesis
206
mitochondrial homeostasis
rates of fission versus fusion control number, size and extent of inter-connections
of mitochondrial network (‘organelle homeostasis’)
defects in mitochondrial network correlate with numerous neurodegenerative diseases (e.g., Alzheimer’s, Parkin son’s)
* fission and fusion controlled by distinct protein machineries
207
Mitochondrial fission (3 steps)
1) ER tubules (subdomain MAM) encircle mitochondrion (via change in ER shape) at future fission site and this initiates constriction
2) Drp1(a dynamin related protein 1) recruited from cytoplasm to constriction site and assemble into helices (Drp1 ring) around surface of mitochondrial outer membrane
recruitement of Drp1 mediated by the lipid microdomain (cardio lipin) in mitochondrial outer membrane
3) conformational change in Drp1 ring due to GTP hydrolysis results in further membrane constriction and fission - forming two daughter mitochondrion
208
Drp1
Drp1 = Dynamin-related protein 1 - member of dynamin GTP-binding protein family responsible for scission of other cellular membranes (e.g., clathrin vesicles @ TGN or pm
KEY PLAYER in mitochondrial fission
forms Drp1 ring that via GTP hydrolysis causes the constriction and fission of the mitochondria!
209
Cardiolipin
mitochondrial-specific membrane phospholipid
normally only found in inner membrane - recruited from inner to outer membrane at constriction site during mitochondrial fission
interacts w Drp1 at constriction site
210
lipid microdomain
is like the bringing and concentration of cardiolipin from inner to outer membrane!
this mediates the recruitment of Drp1 in outer mitochondrial membrane
211
Mitofusins
Mfn1/2
integral outer membrane proteins
possess cytoplasmic-facing GTPase domain and long, coiled-coil, protein-protein interaction domain
located on adjacent mitochondria - link together in GTP-dependent manner to form ‘organelle tethering complex’
212
organelle tethering complex (mitochondria)
Mfn1 and Mfn2 located on adjacent mitochondria - link together in GTP-dependent manner to form ‘organelle tethering complex’
213
Bax and Bak
outer mitochondrial membrane proteins that regulate proper Mfn1/2 binding
they prevent self binding - like red with red or blue with blue
214
Phospholipase D
converts cardiolipin into phosphatidic acid
- forms outer membrane lipid microdomains at sites of tethering
215
Phosphatidic acid
is a cone-shaped lipid
Cardiolipin was converted to this lipid (phosphatidic acid) via Phospholipase D
promotes curvature (bending) of outer membrane during Mfn1/2 mediated membrane fusion
216
OPA1
inner membrane fusion mediated by OPA1
integral inner membrane-bound mitofusin (≈ Mfn1/2)
contains intermembrane space-facing GTPase domain
involved in cristae fusion
OPA1 proteins on adjacent inner membranes
interact in GTP-dependent manner to promote membrane fusion
217
prohibitin
ensures that OPA1-mediated fusion occurs
only between ‘different’ inner membranes
prevents ‘self’ fusion of cristae within same mitochondrion
during last step of mitochondrial fusion
218
Mitochondrial fusion (3 steps)
1) outer membrane tethering
involves Mfn1/2
TPase mitofusins - Mfn1 and Mfn2
integral outer membrane proteins
possess cytoplasmic-facing GTPase domain and long, coiled-coil, protein-protein interaction domain located on adjacent mitochondria - link together in GTP-dependent manner to form ‘organelle tethering complex’
* proper Mfn1/2 binding (e.g., prevention of ‘self’ binding) regulated by other mitochondrial outer membrane
proteins - Bak and Bax
2) outer membrane fusion
involves conversion of cardiolipin to phosphatidic acid via phospholipase D
and also Mfn1/2
outer membrane fusion
* formation of outer membrane lipid ‘microdomains’
at sites of Mfn1/2 ‘tethering’
Phospholipase D converts cardiolipin (moves from inner
to outer membrane) into phosphatidic acid
* phosphatidic acid - ‘cone-shaped lipid’
promotes curvature (bending) of outer membrane during
Mfn1/2-mediated membrane fusion (via GTP hydrolysis)
3) inner membrane fusion
involves OPA1 and prohibitin
inner membrane (cristae) fusion
* inner membrane fusion mediated by OPA1 (OPtic Atrophy 1)
integral inner membrane-bound mitofusin (≈ Mfn1/2)
contains intermembrane space-facing GTPase domain
* OPA1 proteins on adjacent inner membranes
interact in GTP-dependent manner to promote
membrane fusion (via GTP hydrolysis)
* OPA1 binding regulated by other mitochondrial
inner membrane proteins
e.g., Prohibitin - ensures that OPA1-mediated fusion occurs
only between ‘different’ inner membranes
prevents ‘self’ fusion of cristae within same mitochondrion
219
mitochondria targeting sequence
all nuclear-encoded mitochondrial proteins possess unique targeting sequences
specific sequences of amino acids - serve as ‘zipcodes’ to mediate protein targeting
i) from cytoplasm to surface of mitochondrion AND
ii) to 1 of 4 specific mitochondrial subcompartment
220
matrix targeting sequence
most matrix-destined proteins possess a 20-50
amino-acid-long matrix targeting sequence
located at nascent protein’s N terminus
consists of amphipathic -helix
enriched in positively-charged residues (R/K) on one side of helix and hydroxylated (S/T) residues on other side
cleaved following protein import into matrix
221
cytoplasmic molecular chaperones
cytosolic Hsp70
recognizes synthesized protein on free ribosome and binds to keep it in an import-competent state
maintain conformation of nascent protein in
partially unfolded, import-competent state
222
mRNA localization
nascent mitochondrial proteins enriched
in vicinity of mitochondria surface due to
diffusion and.....
mRNA localization
They're recruited and binds to Tom 20/22
mRNAs encoding mitochondrial proteins often enriched in cytoplasm surrounding
mitochondria - ‘Mitochondrial RNA cloud
223
Mitochondrial RNA cloud
mRNAs encoding mitochondrial proteins often enriched in cytoplasm surrounding
mitochondria - ‘Mitochondrial RNA cloud’
* mediated by RNA-binding proteins located on mitochondrial outer surface
224
Tom20 /Tom22
at surface of mitochondrion, protein’s matrix - targeting sequence recognized (bound) by
import receptor complex
* consist primarily of two integral outer membrane proteins -Tom20 and Tom22
Translocon of the outer membrane of 20 and 22 kDa
225
general import pore
general import pore in outer membrane
* consists primarily of integral outer membrane protein Tom40
* referred to as “general” import pore
all (most) mitochondrial proteins (both matrix and membrane-bound) access mitochondria initially through TOM40
* Tom40 forms transmembrane channel
226
inner membrane channel
precursor protein transferred through general
import pore and then through adjacent inner
membrane channel
* consists of integral inner membrane proteins
Tim17, Tim23, and Tim44
Translocon of the inner membrane of 17, 23, and 44 kDa
227
membrane contact sites
general import pore and inner membrane channel adjacent to each other at contact sites places where outer and inner membranes are closely appressed –
intermembrane space reduced or absent at contact sites
contact sites maintained by interactions of Tom40 and Tim23/17 intermembrane-space-facing domains
228
matrix Hsp70
emerging precursor protein also recognized and bound by matrix Hsp70
located at matrix-face of inner membrane channel via binding to Tim44
inner membrane channel accessory protein
229
Molecular motor
matrix Hsp70 acts as molecular motor (‘ratchet’)
Tim44-bound Hsp70 undergoes ATP-dependent conformational changes that ‘pulls’ protein into matrix AND prevents ‘back sliding’ of protein back into cytoplasm
230
H+ electrochemical gradient
during protein translocation [Steps 4-5], import driven partially by H+ electrochemical gradient across inner membrane – established during electron
transport
The concentration of H+ in intermembrane space is greater than concentration of H+ in the matrix
[H+] intermembrane space > [H+] matrix
positively-charged residues in amphipathic matrix - targeting sequence are attracted (‘pulled’) to less positively-charged matrix
231
matrix molecular chaperones
imported, cleaved (mature) protein in matrix
folds without further assistance into final,
active conformation [Step 6]
OR
* final folding of imported, cleaved protein in
matrix requires additional matrix-localized
molecular chaperones and ATP hydrolysis
232
Chloroplasts
semi-autonomous plant cell organelle – derived from photosynthetic cyanobacterium
* site of photosynthesis (Ps) - also involved in several other important metabolic processes
* chloroplasts (≈ mitochondria) are often highly mobile
* move along cytoskeleton elements via molecular motors
233
photosynthesis
also involved in several other important metabolic processes
E.g., fatty acid and amino acid biosynthesis, nitrogen and sulfur assimilation
234
Chloroplast envelope
consists of outer and inner membranes
235
outer membrane chloroplast
contains porins, not as permeable
to ions/small molecules as outer
membrane of mitochondria
236
inner membrane chroloplast
highly impermeable, contains various transporters
237
How do outer and inner membranes of chloroplast differ
outer and inner membranes differ in overall
protein and lipid composition – functionally
distinct membranes
outer membrane - contains porins and is more permeable than inner membrane
inner membrane - highly impermeable - contains various transporters
238
thylakoids
3rd (internal) membrane system
flattened membranous discs arranged in stacks
there are grana thylakoids
and
stromal thylakoids
239
grana thylakoids
the pancakes within the stack
240
stroma thylakoids
the flattened disks between stacks
241
thylakoid membranes
site of ATP synthase
maintain H+ gradient in thylakoid lumen
242
thylakoid lumen
aqueous interior of thylakoid
high H+
(thylakoid lumen like mitochondrial intermembrane space)
243
stroma
aqueous interior (space inside of envelope and outside of thylakoid)
contains enzymes involved in carbohydrate synthesis and plastid genome
also contains ribosomes - translation of plastid genome-encoded proteins
244
plastid genome
the type of genome that thylakoid have
circular; size and copy/gene number varies
between plant species encodes ribosomal proteins, some Ps proteins, including a few located in thylakoid, tRNA and rRNAs, and some RNA polymerase subunits
245
stromules
long, stroma-filled membrane tubules – branched & dynamic - rapidly extend and contract
tail-like thing that was recently discovered and links chloroplasts
allows for efficient metabolite transfer, communication, etc. between chloroplast and/or other organelle (e.g., ER, mitochondria, etc)
246
chloroplast fission
chloroplasts multiply by fission – divide (in synchronization) in response to environmental stimuli and/or developmental signals
two sets of protein machineries involved in chloroplast division: FtsZ and PD
* both form ring-like structures either on
inside or outside of chloroplast envelope
rings ‘tighten’ and ‘pinch’ organelle into two
daughter chloroplasts
247
FtsZ division machinery
internal machinery - located on stromal side of inner membrane
derived from cyanobacterial endosymbiont from which chloroplasts evolved
* includes FtsZ1 and FtsZ2
they form Z ring at future chloroplast division site during fission
248
FtsZ1 and FtsZ2
soluble stromal proteins; homologs of bacterial division protein FtsZ
* FtsZ1/2 spontaneously assemble into long, filamentous polymers at midway point (equator) on inner chloroplast membrane surface
* form FtsZ-ring (‘Z-ring’) at future chloroplast division site
initial positioning of FtsZ proteins at equator is mediated by
several other soluble, stromal proteins - ARC3, MinD and MinE
249
ARC3, MinD and MinE
mediate initial positioning of FtsZ proteins at equator is mediated by several other soluble, stromal proteins - ARC3, MinD and MinE
250
ARC 6
helps w chloroplast fission
integral inner membrane spanning protein localized to equator in dividing chloroplast
linkage of Z-ring to inner membrane at chloroplast division site mediated by ARC6
(Accumulation and Replication of Chloroplasts 6)
* integral inner (envelope) membrane-spanning protein localized to equator in dividing chloroplast stromal-facing domain of ARC6 links to and stabilizes FtsZ-ring
mutation in ARC6 gene disrupts fission process – results in formation of 1-2 giant chloroplasts
ARC6 also controls ‘tightening’ of FtsZ-ring – ARC6’s interaction with Z-ring signals start of
chloroplast constriction process
* intermembrane space (IMS)-facing domain of ARC6 binds to Plastid Dividing (PD)
machinery in chloroplast outer membrane
251
Plastid Dividing (PD)
machinery
intermembrane space (IMS)-facing domain of ARC6 binds to Plastid Dividing (PD)
machinery in chloroplast outer membrane
* includes PDV1 and PDV2 (Plastid Division 1 and 2) hetero-dimeric, integral outer transmembrane proteins recruited
(via binding to ARC6) to midway point in dividing chloroplast
* IMS-facing domains of PDV1/2 interact
with IMS-facing domain of ARC6
252
ARC 5
that hexagon thing on outer membrane of chloroplast that forms a ring on outside
PDV1 and PDV2 also bind to ARC5 (Accumulation & Replication of Chloroplasts 5) cytoplasmic-facing domains of PDV1 & PDV2 bind to ARC5 at site of division on chloroplast outer membrane surface
ARC5: cytoplasmic, soluble dynamin-related GTPase protein
* ARC5 assembles into ‘spiral-like’ structures - form PD-ring
* PD-ring wraps around outside of chloroplast – ‘tightening’ of PD-ring
(via GTP hydrolysis) causes constriction of outer envelope
253
Chloroplast protein targeting
all chloroplast proteins possess unique targeting sequences
specific sequences of amino acids that serve as ‘zipcodes’ - mediate protein targeting
i) from cytoplasm to chloroplast surface and ii) to specific subchloroplast compartment
* multiple (6) subchloroplast targeting
pathways
* vary depending on protein’s final
location in chloroplast
outer or inner membrane
intermembrane space
stroma
thylakoid lumen or membrane
* different pathways rely on different
‘subchloroplast’ targeting signals and
shared and/or unique import machinery
254
stromal import sequence
most stromal-destined proteins possess a stromal import sequence
located at protein’s N-terminus
enriched in hydroxylated residues (S/T) and small hydrophobic residues – does not form amphipathic -helix
(unlike mito matrix targeting signal)
cleaved following protein import into stroma
255
TOC complex
at surface of chloroplast, protein’s stromal import sequence recognized (bound) by Toc complex
Translocon of the outer chloroplast membrane
multi-protein complex, includes receptor(s) subunits, transmembrane channel, and various accessory proteins
binding of stromal import sequence to Toc receptor involves GTP (i.e., energy requirement for import)
256
Tic complex
precursor protein transferred through Toc
complex and then through adjacent Tic complex
translocon of the inner chloroplast membrane
Toc and Tic complexes adjacent to each other at contact sites
precursor protein translocation occurs across both membranes sequentially
257
stromal processing enzyme
cleaves stromal-import sequence as it enters stroma through Tic complex
258
Hsp93
Hsp93 acts as molecular motor (‘ratchet’)
undergoes ATP-dependent conformational changes that ‘pulls’ protein into stroma AND prevents ‘back sliding’ of protein back into cytoplasm & assists with protein folding
similar to Hsp70 of mitochondrial matrix
259
molecular ________________ in stroma [+ATP] ensure cleaved, mature protein properly folded
chaperones (Hsp70)
260
thylakoid targeting sequence
precursor protein imported from cytoplasm into stroma as described above [Step 1], except...
removal of stromal-import sequence by stromal protease reveals thylakoid-targeting sequence
261
SRP-dependent import pathway
precursor protein in stroma maintained in partially unfolded, import-competent state by stromal chaperones [+ATP]
* thylakoid-targeting sequence recognized by
chloroplast signal recognition particle (SRP) [Step 2]
* SRP-binds chloroplast SRP receptor [+GTP] on thylakoid membrane and protein translocated into lumen via thylakoid Sec61-like translocon
* thylakoid-targeting sequence removed by thylakoid lumenal protease [Step 3]
* molecular chaperones in thylakoid lumen [+ATP] mediate folding of mature protein
262
pH-dependent pathway
precursor protein fully folded in stroma [Step 2]
* folded protein imported into thylakoid lumen via di-arginine-containing thylakoid targeting sequence and unique receptor/translocon complex at thylakoid membrane [Step 3]
mechanism for how a large, folded protein is translocated across thylakoid membrane not well understood relies on energy from proton (pH) gradient between
stroma and lumen [H+] thylakoid lumen > [H+] stroma
* thylakoid-targeting sequence on mature protein removed by lumenal protease [Step 4]
263
di-arginine containing thylakoid targeting sequence
folded protein imported into thylakoid lumen via di-arginine-containing thylakoid targeting
sequence and unique receptor/translocon complex at thylakoid membrane
264
thylakoid lumenal protease
thylakoid-targeting sequence on mature protein removed by lumenal protease
265
Thylakoid Sec-61-like translocon
in SRP dependent pathway for targeting proteins to the thylakoid lumen
SRP-binds chloroplast SRP receptor [+GTP] on thylakoid membrane and protein translocated into lumen via thylakoid Sec61-like translocon
266
Chloroplast SRP and SRP receptor
thylakoid-targeting sequence recognized by
chloroplast signal recognition particle (SRP) [Step 2]
* SRP-binds chloroplast SRP receptor [+GTP] on thylakoid membrane and protein translocated into lumen via thylakoid Sec61-like translocon
267
Tunicamycin
a drug that blocks the first step of N-linked glycosylation
