Lecture 8: Intracellular Compartments and Protein Sorting Flashcards
3 features of cellular compartmentalization?
-segregation of metabolic function -organelle-specific environments -membrane surfaces
Segregation of metabolic function
-glycolosis in cytoplasm -Krebs cycle in mitochondria
Organelle-specific environments
-redox potential in ER -changing PH in ER -acidic PH of lysosomes -glycosylation reactions in the ER and golgi
Membrane surfaces
-lipid synthesis -PH gradients and ATP production
What are the three types of transport between compartments?
-gated transport -transmembrane transport -vesicular transport
Identify the type of transport in red
gated transport
Identify the type of transport in blue
Transmembrane transport
Identify the type of transport in green
vesicular transport
How does a protein “know” where to go?
Specific elements of a protein, mostly primary amino acid sequence, direct protein traffic
– Signal sequences
– Signal patches
What signal sequence is this?
import into nucleus
What signal sequence is this?
Return to ER
What signal sequence is this?
Import into mitochondria
What signal sequence is this?
Import into plasmid
What signal sequence is this?
import into peroxisomes
What signal sequence is this?
Import into ER
What signal sequence is this?
Return to ER
Gated transport occurs___
from the cytoplasm to the nucleus
The nuclear envelope is directly connected to the ___
ER
Nuclear pore complex
>125 MegaDaltons
>30 proteins (nucleoporins)
~3,000-4,000 NPCs per nucleus
• Molecules less than ____ Da freely diffuse through the NPC
5,000
Protein between _____ Da diffuse through more slowly proportional to their size
20-60,000
Proteins greater than ___ Da require help to enter
60,000
How do “big” proteins enter and exit the nucleus?
• They use a regulated (gated) pathway that relies on specific import signals or nuclear localization signals
• Nuclear localization signals (NLSs) bind to…
specific receptor proteins that direct import into the nucleus through the NPC
The nuclear localization signal (NLS) was identified by systematically deleting or modifying sequences in _____
SV40 T-antigen
A change as small as ___ can disrupt the activity of an NLS
one amino acid
Functionality as an NLS is ___ its location within the polypeptide
independent of
___ bind NLSs and nucleoporins
Nuclear import receptors
Nuclear import receptors are a family of related protein called ____ or ____
importins or karyopherins
These protein bind “cargo” via the NLS in the cytosol and then bind to the NPC
nuclear import receptors (importins)
• Specific nucleoporins form tentacle-like fibril composed of amino acid repeats ___
- FG (Phenylalanine-Glycine)
- Importins bind to these FG repeats
Different families of import receptors recognize different ____
NLSs (nuclear localization signals)
Some cargos require an ___ that contains the NLS
adapter protein
• Nucleocytoplasmic transport is ____
bidirectional
• Large molecules like _____ must exit the nucleus and enter the cytoplasm through the NPC
new ribosomal subunits and RNA molecules
• Nuclear export is very similar to import, but topologically reversed….
- Depends on nuclear export signals (NES)
- Depends on export receptors (exportins/ karyopherins)
Ran GTPase
A small G-protein that provides energy and directionality to nucleocytoplasmic transport
Like all small G-proteins, ___ is a molecular switch that changes activity based on a bound ____
Ran, nucleotide
• The gradient of the two conformational forms of Ran drive ____ in the appropriate direction
nuclear transport
The Ran GTPase cycle
How Ran provides directionality
RanGTP cargo displacement
Ran-GTP and cargo binding to an import receptor is…
mutually exclusive
The nuclear envelope disassembles during mitosis
The ER network composed of sheets and tubules is regulated by ___
fusion and fission
Posttranslational transmembrane transport
- Mitochondria
- Chloroplasts
- Peroxisomes
Mitochondrial function
- Energy (ATP) production by oxidative phosphorylation
- Intermediary metabolism (TCA cycle)
- Lipid metabolism
– Catabolism (beta-oxidation)
– Synthesis
While mitochondria contain DNA and protein synthesis machinery, the majority of mitochondrial proteins are _____ encoded genes that are postranslationally imported
nuclear
___ and ____ regulate mitochondrial shape
Fusion and fission
Mitochondrial matrix targeting sequence
- 20-50 amino acids
- Rich in hydrophobic, positively charged basic (R,K) and hydroxylated (S,T) amino acids
- Tend to lack acidic residues (D,E)
- Form amphipathic alphahelices
- Cleaved off the mature protein by a matrix protease
Translocation into the mitochondrial matrix depends on ____
a signal sequence and protein translocators
In mitochondrial protein imports, precursor proteins are imported as ____
unfolded polypeptide chains
Proteins are imported into the matrix at ___ that join the inner and outer membrane
contact sites
___ and ____ are used to drive import into mitochondria
ATP hydrolysis and a proton gradient
Repeated cycles of ATP hydrolysis by ___ complete the import process
mHSP70
Transport to the inner membrane and inner membrane space requires ___ signals
two
• Translocase of the Outer Membrane (TOM)
- Required for the import of all nuclear encoded genes
- Helps insert membrane proteins into the outer membrane
Sorting and Assembly Machine (SAM) complex
– Helps insert and fold beta-barrel proteins
What does TIM stand for?
• Translocase of the Inner Membrane (TIM)
TIM23 complex
• Spans both membranes
• Mediates translocation of proteins in to the matrix space and inner membrane proteins
TIM22 complex
Mediates the insertion of a subclass of inner membrane proteins
OXA complex
• Mediates the insertion of inner membrane proteins synthesized in the mitochondrial matrix
Protein translocators in the mitochondrial membrane
Which protein translocaters are in the outer mitochondrial membrane?
- TOM complex
- SAM complex
Which protein translocaters are in the inner mitochondrial membrane?
- TIM22 complex
- TIM23 complex
- OXA complex
The negative membrane potential helps “attract” the_____ charged mitochindrial targeting signal
positively
The integration of Porins (Beta-barrel proteins) into the outer mitochondrial membrane is accomplished by what protein translocators?
TOM and SAM
Protein import into the mitochondrial matrix is accomplished by which two translocators?
TOM and TIM23
How are proteins targeted to the inner mitochondrial membrane?
TOM/TIM23 path but with a stop transfer sequence
OR
TOM/TIM23 path and OXA complex
How are proteins targeted to the inner membrane space of the mitochondria?
TOM/TIM23/OXA then protease cleaves the second signal sequence
OR
TOM/TIM23 and stop transfer sequence, protease cleaves stop transfer sequence
How do you target to the inner membrane space through the outer membrane?
TOM complex, then protein oxidized by Mia40
What are the two complexes responsible for protein import into the stroma of the chloroplast?
TIC (inner membrane)
TOC (outer membrane)
What are the four ways to translocate into the thylakoid space?
Sec pathway
SRP-like pathway
TAT pathway
spontaneous insertion
Peroxisomal Functions
• House oxidative enzymes
– Oxidases produce peroxide (H202), hence the name peroxisome
– Contains catalase to degrade H202
• Lipid metabolism
– fatty acids beta oxidation
- Exclusively peroxisomal in yeast and plants
- Shared with mitochondria in animal
_____ do not contain DNA and all proteins are imported posttranslationally
peroxisomes
At least ___ peroxisomal targeting sequences (PTS) are known for matrix proteins
two
____ import is fundamentally different from other organelles since fully-folded proteins can be imported
peroxisome
___ is a specific protein that catalyzes protein import into peroxisomes
Pex19
___ genes or ___, are proteins responsible for the generation and maintenance of peroxisomes
Pex genes or peroxins
