Post on 04-Jun-2018
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
1/59
Intracellular Compartments
and Protein Sorting
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
2/59
Intracellular Compartments andProtein Sorting
Functionally distinct membrane bound organelles
10 billion proteins of 10,000-20,00 diff kinds
Complex delivery system
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
3/59
Compartmentalization of Cells
Membranes Partition cell Important cellular functions Impermeable to most hydrophobic molecules contain transport proteins to import and export specific molecules Mechanism for importing and incorporating organelle specific proteins
that define major organelles
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
4/59
Compartmentalization of Cells
All Eucaryotic Cells Have Same Basic Set of Membrane Bound Organelles
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
5/59
Compartmentalization of Cells
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
6/59
Compartmentalization of Cells
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
7/59
Compartmentalization of Cells
Major Organelles Nucleus
Cytosol
ER Golgi Apparatus
Mitochondria and Chloroplast
Lysosomes
Endosomes
Peroxisomes
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
8/59
Compartmentalization of Cells
Occupy 50% cell volume
Perform same basic function
Vary in size and abundance
May take on additional functions
Position dictated by cytoskeleton
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
9/59
Compartmentalization of Cells
Topology governed by evolutionary originsInvagination of pm creates organelles such as nucleus that aretopologically equivalent to cytosol and communicate via pores
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
10/59
Compartmentalization of Cells
Topology governed by evolutionary originsEndosymbiosis of mito and plastids creates doublemembrane organelle (have own genome)
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
11/59
Compartmentalization of Cells
Topology governed by evolutionary originsOrganelles arising from pinching off of pm have interiorequivalent to exterior of cell
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
12/59
Compartmentalization of Cells
3 Types of Transport Mechanisms
1. Gated Transport:
gated channels
topologically equivalent spaces2. Transmembrane Transport:
protein translocators
topologically distinct space
3. Vesicular transport:membrane enclosed intermediatestopologically equivalent spaces
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
13/59
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
14/59
Compartmentalization of Cells
2 Types of Sorting Signals in Proteins1. Signal Sequence
continuous sequence of 15-60 aa
sometimes removed from finished protein
sometimes a part of finished protein2. Signal Patch
specific 3d arrangement of atoms on protein surface; aas distant
persist in finished protein
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
15/59
Compartmentalization of Cells
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
16/59
Signal Sequences/PatchesDirect Proteins to Final Destination
Signal patches direct proteins to:1. nucleus2. lysosomes
Signal Sequences direct proteins to:
1. ER proteins possess N-terminal signal of 5-10 hydrophobic aa2. mito proteins have alternating + chg aa w/ hydrophobic aa3. proxisomal proteins have 3 aa at C-terminus
Compartmentalization of Cells
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
17/59
Compartmentalization of Cells
Sorting signals recognize complementary sorting receptors Receptors unload cargo Function catalytically and are reusable
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
18/59
Compartmentalization of Cells
Organelles Cannot be Constructed Denovo
Organelles reproduced via binary fission Organelle cannot be reconstructed from DNA alone Info in form of one protein that pre-exists in organelle mem is required
and passed on from parent to progeny Epigenetic information essential for propogation of cells compartmental
organization
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
19/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Envelope Two concentric membranes
-Outer membrane contiguous w/ER-Inner membrane contains proteins thatact as
binding sites for chromatin and nuclearlamina
Perforated by nuclear pores for selectiveimport and export
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
20/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Pore Complex mass of 125 million; ~50 differentproteins arranged in octagon
Typical mammalian cell 3,000-4,000 Contains >1 aqueous channels thruwhich sm molec can readily pass 60,000 cannot pass
Functions ~diaphram Receptor proteins actively transportmolec thru nuclear pore
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
21/59
Transport of Molecules Btwn Nucleus and Cytosol
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
22/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Localization Signal Generally comprised of two short sequences rich in + chged aa lys & arg Can be located anywhere Thought to form loops or patches on protein surface Resident, not cleaved
Transport thru lg aqueous pores as opposed to translocator proteins Transports proteins in folded state Energy requiring process
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
23/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Import- the players Importins = cytosolic receptor protein binds to NLS of cargo proteins Nucelar Export Receptors = binds macromolecules to be exported from nucelus Adaptors = sometimes required to bind target protein to nuclear receptor Ran = cytosolic GTP/GDP binding protein complexes with importins in the cytosol. Fibril proteins and nucleoporins contain phenylalanine/glycine repeats (FG) repeats.
Repeats transiently bound and released by importin/cargo/Ran-GDP, causing thecomplex to hop into the nucleus
Import Receptors release cargo in nucleus and return to cytosol
Export Receptors release cargo in cytoplasm and return to nucleus
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
24/59
Transport of Molecules Btwn Nucleus and CytosolRan GTPase= molecular switch
Drives directional transport in appropriate directin Conversion btwn GTP and GDP bound states mediated by Ran specific regulatory proteinsGAP converts RNA-GTP to Ran-GDP via GTP hydrolysisGEF promotes exchg of GDP for GTP converting Ran-GDP to Ran-GTP
Ran GAP in cytosol thus more Ran-GDP in cytosol Ran GEF in nucleus thus more Ran-GTP in nucleus
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
25/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Export Works like import in reverse
Export receptors bind export signals and nucleoporins to guidecargo thru pore Import and export receptors member of same gene family
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
26/59
Transport of Molecules Btwn Nucleus and Cytosol
Regulation Afforded by Access to Transport Machinery Controlling rates of import and export determines steady state location phosphorylation/dephosphorylation of adjacent aa may be required for receptor binding Cytosolic anchor or mask proteins block interaction w/ receptors Protein made and stored in inactive form as ER transmembrane protein
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
27/59
Transport of Molecules Btwn Nucleus and Cytosol
Control of mRNA Export Proteins w/ export signals loaded onto RNA during transcription and
processing (RNP) Export signals guide RNA out of nucleus thru pores via exportin proteins than
bind RNP
Export mediated by transient binding to FG repeats Imature mRNAs retained by anchoring to transcription and splicing machinery Proteins disassociate in cytosol and return to nucleus
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
28/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear Lamina Meshwork of intermediate filaments
Maintenance of nuclear shape
Spacial organization of nuclear pores
Regulation of transcription
Anchoring of interphase chromatin
DNA replication
Phosphorylation causes depolymerizesduring mitosis when nucleus disassembles
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
29/59
Transport of Molecules Btwn Nucleus and Cytosol
Nuclear envelop disassembles during mitosis and reassembles
when ER wraps around chromosomes and begins to Fuse
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
30/59
Protein Transport into theMitochondria and Chloroplast
Subcompartments of the Mitochondria and Chloroplast
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
31/59
Protein Transport into theMitochondria and Chloroplast
Translocation into Mitochondrial Matrix Governed by: 1. Signal Sequence (amphipathic alpha helix cleaved after import)
2. Protein Translocators
P i T i h
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
32/59
Protein Transport into theMitochondria and Chloroplast
Players in Protein Translocation of Proteins in Mitochondria TOM- functions across outer membrane TIM- functions across inner membrane OXA- mediates insertion of IM proteins syn w/in mito and helps to
insert proteins initially transported into matrix
Complexes contain components that act as receptors andothers that form translocation channels
P i T i h
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
33/59
Protein Transport into theMitochondria and Chloroplast
Import of Mitochondrial Proteins
Post-translational Unfolded polypeptide chain
1. precursor proteins bind to receptor proteins of TOM
2. interacting proteins removed and unfolded polypetide is fed into
translocation channel Occurs contact sites joining IM and OM
TOM transports mito targeting signal across OM and once it reaches IMtargeting signal binds to TIM, opening channel complex thru which proteinenters matrix or inserts into IM
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
34/59
Protein Transport into theMitochondria and Chloroplast
Import of Mitochondrial Proteins Requires energy in form of ATP and H+ gradient and assitance of hsp70
-release of unfolded proteins from hsp70 requires ATP hydrolysis
-once thru TOM and bound to TIM, translocation thru TIM requires
electrochemical gradient
P i T i h
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
35/59
Protein Transport into theMitochondria and Chloroplast
Protein Transport into IM or IM Space Requires 2 Signal Sequences
1. Second signal =hydrophobic sequence; immediately after 1st signal sequence
2. Cleavage of N-terminal sequence unmasks 2 nd signal used to translocate proteinfrom matrix into or across IM using OXA
3. OXA also used to transport proteins encoded in mito into IM
4. Alternative route bypasses matrix; hydrophobic signal sequence = stoptransfer
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
36/59
Protein Transport into theMitochondria and Chloroplast
Protein Transport into Chloro Similar to Transport into Mito1. occur posttranslationally2. Use separate translocation complexes in ea membrane3. Translocation occurs at contact sites4. Requires energy and electrochemical gradient5. Use amphilpathic N-terminal signal seq that is removed6. Like the mito a second signal sequence required for translocation
into thylakoid mem or space
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
37/59
Protein Transport into theMitochondria and Chloroplast
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
38/59
Peroxisomes and Protein Import
Peroxisomes Use O 2 and H 2O2 to carry out oxidative rxns Remove H from specific organic compounds RH 2 + O 2 R + H 2O2 Catalases use H 2O2 to oxidize other substances, particularly in liver and kidney detoxification
H2O2 + RH 2 R + H 2O Beta Oxidation Formation of plasmalogens (abundant class of phospholipids in myelin) Photorespiration and glyoxylate cycle in plants
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
39/59
Peroxisomes and Protein Import
Peroximsomes in Plants
Site of Photorespiration= glycolate pathway in leaves Called glyoxysomes in seeds where fats converted intosugar
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
40/59
Proxisomes and Protein Import Peroxisomes arise from pre-existing peroxisomes Signal sequence of 3 aa at COOH end of peroxisomal proteins= import signal Some have signal sequence at N-terminus Involves >23 distinct proteins Driven by ATP hydrolysis Import mechanism distinct, not fully characterized Oligomeric proteins do not unfold when imported Zellweger Disease= peroxisomal deficiency
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
41/59
ER and Protein Trafficking
Endoplasmic Reticulum Occupies >= 50% of cell volume Continuous with nuclear membrane Central to biosyn macromolecules used to construct other organelles Trafficking of proteins to ER lumen, Gogli, lysosome or those to be secreted
from cell
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
42/59
ER and Protein Trafficking
ER Central to Protein Synthesis and Trafficking Removes 2 Types of Proteins from Cytosol:
1. transmembrane proteins partly translocated across ER embedded in it
2. water soluble proteins translocated into lumen
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
43/59
ER and Protein Trafficking
Quantity of SER and ER Dependent Upon Cell TypeRER assoc. w/ protein synthesis
SER assoc. lipid biosynthesis, detoxification, steroid synthesis, Ca2+
storage
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
44/59
ER and Protein Trafficking
Import of Proteins into ER Occurs co-translationally
Signal recognition sequence recognized by SRP
SRP recognized by SRP receptor
Protein Translocator
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
45/59
ER and Protein Trafficking
Hydrophobic signal sequence of diff sequence and shape SRP lg hydrophobic pocket lined by Met having unbranched flexible
side chains Binding of SRP causes pause in protein synthesis allowing time for
SRP-ribosome complex to bind to SRP receptor
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
46/59
ER and Protein Trafficking
Protein to be imported passes through an aqueous pore in
the translocator that is a dynamic structure Sec61 protein translocator
Signal sequence triggers opening of pore
Translocator pore closes when ribo not present
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
47/59
ER and Protein Trafficking
Some proteins are imported in to ER by a posttranslational mechanism
Proteins released into cytoplasm Binding of chaperone proteins prevents them from folding
Translocation occurs w/out ribo sealing pore
Mechanism whereby protein moves through pore unkwn
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
48/59
ER and Protein Trafficking
Signal Sequence is Removed from Soluble Proteins Two signaling functions:
1) directs protein to ER membrane2) serves as start transfer signal to open pore
Signal peptidase removes terminal ER signal sequence uponrelease of protein into the lumen
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
49/59
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
50/59
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
51/59
ER and Protein Trafficking
Folding of ER Resident Proteins ER resident proteins contain an ER
retention signal of 4 specific aa at C-terminus
PDI protein disulfide isomerase oxidizesfree SH grps on cysteines to from disulfidebonds S-S allowing proteins to refold
BiP chaperone proteins, pulls proteinsposttranslationally into ER thru translocator
and assists w/ protein folding
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
52/59
ER and Protein TraffickingGlycolsylation of ER Proteins Most soluble and transmembrane proteins made in ER are
glycolsylated by addition of an oligosaccharide to Asn Precursor oligosaccharide linked to dolichol lipid in ER mem, in
high energy state
Transfer by oligosaccharyl transferase occurs almost as soon aspolypeptide enters lumen
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
53/59
ER and Protein Trafficking
Oligosaccharide assembled sugar by sugar onto carrier lipid dolichol
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
54/59
ER and Protein Trafficking
Retrotranslocation Improperly folded ER proteins are exported and degraded in cytosol Misfolded proteins in ER activate an Unfolded Protein Response to
increase transcription of ER chaperones and degradative enzymes
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
55/59
ER and Protein Trafficking
The Unfolded Protein Response
ff
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
56/59
ER and Protein Trafficking Assembly of Lipid Bilayers on ER
ER synthesizes nearly all major classes of lipids Phospholipid synthesis occurs on cytoplasmic face by enzymes in
mem Acyl transferases add two FA to glycerol phosphate producing
phosphatidic acid
Later steps determine head group
ER d P t i T ffi ki g
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
57/59
ER and Protein Trafficking
Assembly of Lipid Bilayers on ER Scramblase phospholipid translocator equilibrates phospholipids
distribution Flipasses of PM responsible for asymmetric distribution of phospholipids
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
58/59
8/13/2019 Chpt 12 Intracellular Compartments anddfd Protein Sorting
59/59
Transport of Molecules Btwn Nucleus and Cytosol