Cell Biology (BIO 320)
Chapter 12 – Intracellular compartments and protein sorting
I.
II.
III.
IV.
V.
The compartmentalization of cells (pages 695-704; figures 12-1 to 12-7; tables 12-1 to 12-3)
The transport of molecules between the nucleus and the cytosol (pages 704-712; figures 12-8 to
12-20)
The transport of proteins into mitochondria and chloroplasts (pages 713-720; figures 12-21 to
12-28)
Peroxisomes (pages 721-723; figures 12-30 to 12-33)
The endoplasmic reticulum (pages 723-745; figures 12-34 to 12-58)
I
1.
The compartmentalization of cells
All eucaryotic cells have the same basic set of membrane-enclosed compartments –
function of each compartment; their contribution to the total cell volume; relative
amounts of membrane contributed by each compartment.
The plasma membrane is only a minor membrane in most eucaryotic cells; in most cells
the ER membrane and the mitochondrial inner membrane are the most abundant; what
are the functions performed by these membranes?
2.
Toplogical relationships between different membrane-enclosed compartments can be
interpreted from their evolutionary origins.
The intracellular compartments in eucaryotic cells can be grouped into four distinct
families based on their evolutionary origins:
(i)
the nucleus and the cytosol
(ii)
ER, Golgi apparatus, endosomes, lysosomes, transport vesicles, and possibly
peroxisomes
(iii) mitochondria
(iv)
plastids (in plants only)
3.
Proteins can move between compartments in different ways
(i)
Gated transport
(ii)
Transmembrane transport
(iii) Vesicular transport
4.
Signal sequences and signal patches direct proteins to the correct cellular address.
5.
Most organelles are generated by growth and division of existing organelles.
II.
1.
The transport of molecules between the nucleus and the cytosol
Nuclear pore complexes – their structure and the size of molecules that can be transported
through them. Bidirectional traffic occurs continuously between the cytosol and the
nucleus.
2.
Nuclear localization signals direct nuclear proteins to the nucleus. Fully folded proteins
synthesized in the cytosol are recognized and transported across the nuclear envelope
through nuclear pores.
3.
Nuclear import receptors bind nuclear localization signals and NPC proteins
(nucleoporins.)
The majority of nucleocytoplasmic transport cargoes are recognized by a family of
soluble transport factors called importins, exportins or transportins, and collectively
referred to as karyopherins. The interactions between karyopherins and the NPC occur
largely through several nucleoporins containing so-called 'FG domains'.
4.
Nuclear export works like nuclear import, but in reverse.
5.
The Ran GTPase drives directional transport through nuclear pore complexes
Passage through the pore in either direction seems to be independent of metabolic energy.
However, once on the target side, transport is terminated by the asymmetric distribution
of the GTP- and GDP-bound forms of the GTPase Ran. The Ran-GTP form is
maintained predominantly in the nucleus, where it breaks apart import complexes while
stabilizing export complexes. Conversely, hydrolysis of GTP in the cytoplasm produces
Ran-GDP and triggers the release of export cargo. It is generally considered that the
directionality of transport is conferred, in whole or at least in part, by this differential
localization of the two Ran species.
6.
Transport between nucleus and the cytosol can be regulated by controlling access to the
transport machinery.
7.
The nuclear envelope is disassembled during mitosis.
III.
The transport of proteins into mitochondria and chloroplasts
1.
Nature (soluble or membrane) and functions of proteins transported from the cytosol to
the mitochondria and chloroplasts.
2.
The different compartments of a mitochondrion – outer membrane, intermembrane space,
inner membrane, and matrix space – examples of proteins in these compartments and
their functions.
3.
The different compartments of a chloroplast – outer membrane, intermembrane space,
inner membrane, stroma, thylakoid membrane, and the thylakoid space – examples of
proteins in these compartments and their functions.
4.
Translocation of protein from the cytosol into the mitochondrial matrix – posttranslational; N-terminal cleavable signal sequence that forms an amphipathic helix;
protein tranlocators on the mitochondrial membranes.
5.
The properties of a signal sequence for mitochondrial protein import.
6.
The protein translocators in the mitochondrial membranes:
The TOM complex is required for the import of all nucleus-encoded mitochondrial
proteins.
The TIM23 complex transports some of these proteins into the matrix space; helps insert
transmembrane proteins into the inner membrane.
The TIM22 complex mediates insertion of a subclass of inner membrane proteins.
The OXA complex mediates insertion of inner membrane proteins synthesized in the
mitochondria.
The SAM complex helps the folding of -barrel proteins in the outer membrane.
7.
Mitochondrial precursor proteins are imported as unfolded polypeptide chains. How are
these polypeptide chains kept in an unfolded state following their synthesis in the
cytosol?
8.
Steps in the import of proteins from the cytosol to the mitochondrial matrix.
The TOM complex first transports the signal sequence across the outer membrane to the
intermembrane space, where it binds to a TIM complex, opening the channel in the
complex. The polypeptide chain then either enters the matrix space or inserts into the
inner membrane.
9.
ATP hydrolysis and a H+ gradient are required for protein import into mitochondria
hsp70 and ATP in the cytosol is not required if the precursor protein is artificially
unfolded prior to adding to purified mitochondria.
10.
Protein transport into the inner mitochondrial membrane and the intermembrane space
requires two signal sequences.
11.
Arrangement of targeting sequences in imported mitochondrial proteins.
12.
Two signal sequences are required to direct proteins to the thylakoid membrane in
chloroplasts.
13.
Four possible pathways for transporting proteins from the cytosol to the thylakoid space.
IV.
1.
Peroxisomes
Peroxisomes are found in all eucaryotic cells and are major sites of oxygen utilization.
They contain one or more enzymes that use molecular oxygen to remove hydrogen atoms
from organic compounds and produce H2O2 in the reaction
RH2 + O2 → R + H2O2
Catalase utilizes the hydrogen peroxide to oxidize a variety of other substrates by the
“peroxidative” reaction detoxifying various toxic molecules.
2.
Steps in the import of peroxisomal matrix proteins directed by PTS1 targeting sequence
Step 1: Catalase and most other peroxisomal matrix proteins contain a C-terminal PTS1
uptake-targeting sequence (-Ser-Lys-Leu-COO¯ or a related sequence) that binds to the
cytosolic receptor Pex5. PTS2 is an amino-terminal targeting sequence contained in a
protein leader sequence, which is cleaved (?) by a peroxisome-specific protease after
import of the proteins into the matrix. Peroxisomal 3-ketoacyl-CoA thiolases are the best
example of proteins with PTS2 sequences. The PTS2 consensus sequence in plants is: R
XXX (I/L) XXHL.
Step 2:Pex5 with the bound matrix protein interacts with the Pex14 receptor located on
the peroxisome membrane.
Step 3:The matrix protein-Pex5 complex is then transferred to a set of membrane
proteins that are necessary for translocation into the peroxisomal matrix by an unknown
mechanism. Translocation across the peroxisomal membrane depends on ATP
hydrolysis and the proteins traverse the membrane in a folded conformation.
Step 4:At some point, either during translocation or in the lumen, Pex5 dissociates from
the matrix protein and returns to the cytosol.
V.
The Endoplasmic Reticulum (ER)
1.
The ER has a central role in lipid and protein synthesis.
2.
Membrane-bound ribosomes define the rough ER. The ER captures transmembrane and
selected water-soluble proteins from the cytosol as they are being synthesized.
3.
The smooth ER, sometimes called transitional ER contain exit sites from which transport
vesicles carrying newly synthesized proteins and lipids bud off for transport to the Golgi
apparatus.
4.
Smooth ER is abundant in some specialized cells where it performs additional functions.
For example, cells that synthesize steroid hormones from cholesterol have an expanded
smooth ER compartment.
5.
A common pool of ribosomes is used to synthesize the proteins that complete their
synthesis in the cytosol and those that complete their synthesis on the membrane of the
ER.
6.
Rough and smooth regions of ER can be separated by centrifugation.
7.
Signal sequences were first discovered in proteins imported into the rough ER.
8.
A Signal-Recognition Particle (SRP) directs ER signal sequences to a specific receptor in
the rough ER membrane.
9.
How do ER signal sequences and SRP direct ribosomes to the ER membrane?
10.
The SRP receptor in the ER membrane is composed of two different polypeptides.
11.
A model for SRβ-mediated regulation of SRP-dependent protein targeting to the
endoplasmic reticulum. SRP binds to the signal sequence, emerging from the ribosome
exit site via SRP54, and protein translation is delayed. This ribosome-nascent chain
(RNC)-SRP complex is targeted to the ER membrane by SR. SRβ (cyan) resides in the
membrane and has to be loaded with GTP by an exchange factor in order to bind SRα
(binding domain SRX in magenta). The exchange factor for SRβ is not known, and the
translocon is a potential candidate. In the assembled SR-SRP-RNC complex, all three
GTPases are loaded with GTP and the complex is now destined for translocation. The
signal sequence is then transferred to the translocon, SRP and SR dissociate, and protein
elongation resumes. GTP hydrolysis of SRP54, SRα, and SRβ can occur after nascent
chain transfer in a yet poorly understood fashion.
(Schwartz and Blobel, 2003, Cell 112:793-803)
12.
The polypeptide chain passes through an aqueous pore in the translocator.
13.
Three ways in which protein translocation can be driven through structurally similar
membranes.
14.
The ER signal sequence is removed from most soluble proteins after translocation.
15.
Integration into the ER membrane of a single-pass transmembrane protein with a cleaved
signal sequence.
16.
Integration into the ER membrane of a single-pass transmembrane proteins with an
internal signal sequence.
17.
Integration into the ER membrane of a double-pass transmembrane protein with an
internal signal sequence.
18.
Integration into the ER membrane of a multipass transmembrane protein.
19.
Examples of translocated polypeptide chains that fold and assemble in the lumen of the
rough ER.
20.
Most proteins synthesized in the rough ER membrane are glycosylated by the addition of
a common N-linked oligosaccharide. A sequence of amino acids (--Asn - X - Ser/Thr --)
in the translocated region of the polypeptide is recognized by the membrane-bound
enzyme oligosaccharyl transferase that attaches the oligosaccharide to the asparagine side
chain. What are the other kinds of protein glycosylation? Where do they occur?
21.
Oligosaccharides are used as tags to mark the state of protein folding. The glucosyl
transferase determines whether the protein is folded properly or not.
22.
Improperly folded proteins are exported from the ER and degraded in the cytosol.
23.
Misfolded proteins in the ER activate an unfolded protein response.
24.
Some membrane proteins acquire a covalently attached GPI anchor.
25.
The ER assembles most lipid bilayers.