Intracellular Compartments And Protein Sorting

Front 1

Basic set of membrane-enclosed-compartments

Back 1

cytosol~54%
mitochondria~22%
Rough ER~9%
Smooth ER~6%
Nucleus~6%
Peroxisomes~1%
Lysosomes~ 1%
Endosomes~1%

Front 2

cytosol

Back 2

about 50%
site of protein synthesis and degradation
performs most of the cells intermediate metabolism

Front 3

Mitochondria

Back 3

about 22%
generate most ATP that cell uses

Front 4

peroxisomes

Back 4

about 1%
contain enzymes used in various oxidation reactions

Front 5

nucleus equivalent to

Back 5

cytosol

Front 6

ER equivalent to

Back 6

golgi apparatus

Front 7

mitochondria equivalent to

Back 7

none

Front 8

plastids equivalent to

Back 8

none

Front 9

proteins can move between compartments in different ways

Back 9

gated transport
transmembrane transport
vesicular transport

Front 10

most organelles are generated by

Back 10

growth and division of existing organelles

Front 11

3 main ways proteins can move into membrane bound compartments

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1)gated transport
2)transmembrane transport
3) vesicular transport

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gated transport

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transport into and out of the nucleus

Front 13

transmembrane transport

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import into ER, mitochondria, plastids, peroxisome

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vesicular transport

Back 14

transport between ER, golgi, outermembrane

Front 15

2 types of signal

Back 15

signal sequences- continous stretched of aas that are recognized when folded
signal patches-stretches of aas that arent recognized by as signals until folded

Front 16

general properties of signal sequence to ER

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N-term, removal of sequence, core of 6-12 hydrophobic amino acids

Front 17

general properties of signal sequence mitochondria

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N-term, removal of sequence, amphipathic helix, Arg and Lys residues on one side, hydrophobic residues on other

Front 18

general properties of signal sequence to choloroplast

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N-term, removal of sequence, no common motifs

Front 19

general properties of signal sequence to peroxiomes

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C-TERM, NO removal of sequence,PTS1 signal at C-term PTS2 at N-term

Front 20

antigen is recognized by

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t-cells

Front 21

calcineuron gets activated when bound to

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Calcium

Front 22

How is the signal exposed

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Calcineuron cleaves NF-AT

Front 23

dissassembly of nuclear envelope due to

Back 23

phospohrylation

Front 24

TOM complex is required for

Back 24

import of ALL nucleus-enclosed mitochonrial proteins
on outer membrane

Front 25

The TIM23 complex

Back 25

transports some of these proteins into the matrix space, helps insert transmembrane proteins into inner membrane

Front 26

TIM 22 complex

Back 26

mediates insertion of a subclass of inner membrane proteins

Front 27

OXA complex

Back 27

mediates the insertion of inner membrane proteins synthesized in the mitochondria

Front 28

mitochondrial precursor proteins are imported as

Back 28

unfolded polypeptide chains

Front 29

T/F like the lumen of the ER, the interior of the nucleuys is topologically equivalent to the outside of the cell

Back 29

false. flow of material does not have to cross a bilayer in nucleus but does in ER

Front 30

membrane-bound and free ribosomes, which are structureally and functionally identical, differ only in the proteins they happen to be making at a particular time

Back 30

true. ribosomes all begin translating mRNAs in cytosol

Front 31

to avoid the inevitable congestion that would occur if two-way traffic through a single pore were allowed, nuclear pore complexes are specialized so that some mediate import while others mediate export

Back 31

false. Individual nuclear pores mediate in both directions.

Front 32

peroxisomes are found only in a few specialized types of eukaryotic cells

Back 32

false, found in all

Front 33

in multipass membrane proteins, the odd numbered transmembrane segments act as start-transfer signals and the even numbered act as stop-transfer signals

Back 33

False. the most N-terminal acts as a start transfer signal

Front 34

what is the fate of a protein with no sorting signal?

Back 34

it will remain in the cytosol

Front 35

imagine you have engineered a set of genes, each encoding a protein with a pair of conflicting signal sequences that specify different compartments. If the genes were expressed in a cell, predict which signal would win out for the following combos

Back 35

A. Signal for import to nuclues and import to ER. The protein would enter the ER. The signal for import to ER is located at the N-term and functions before the internal signal for nuclear import is synthesized.
B. Signals for import into peroxisomes and import to ER. Import to ER is located at N-term and functions before the internal signal for peroxisomes is synthesized.
C. signals for import into mitochondria and retention in ER. Protein would enter mitochondira because in order for retention in ER, protein must be imported into ER but there is no ER import signal
D. Signals for import into nucleus and export from nucleus. It would shuttle between the cytosol and nucleus. These signals do not really conflict

Front 36

What portion of the nucleoplasmin molecule is respobsible for localization in nucleus?

Back 36

This portion is in the tail.

Front 37

How do these experiments distinguish between active transportm in which a nuclear localization signal triggers transport by the nuclear pore complex, and passive diffusion

Back 37

these experiments suggest that the nucleoplasmin tail carries a nuclear localization signal and that accumulation in the nucleus is not the result of passive diffusion. The experiment suggests the heads are too large to pass with passive diffusion

Front 38

Why did you use RANQ69L-GTP instead of RAn GTP?

Back 38

Ran is a GTPase and will slowly convert GTP to GDP and so you would have had an undefined mixture of GTP and GDP which would confuse the results. By using a form of RAN that cannot hydrolize GTP, you are assured that Ran was in Ran-GTP conformation

Front 39

Which of the many proteins eluted from the 2 different affinity columns is likely a candidate for the factor that promotes nuclear import of Ran-GDP

Back 39

since Ran69L-GTP column was not removed, you are looking for a protein that is present in lane 1 but not in lane 2. One such protein is located between 7 kd and 14 kd.

Front 40

what other protein or proteins would you predict that the ran-GDP import factor would be likely to bind in order to carry out its function

Back 40

the small protein that binds to Ran-GDP called NTF2. NTF2 binds to Ran-GDP and FG-repeats present in the nucleoporins of the nuclear pore complex. The progressive movement of NTF2 along FG tracks in nucleoporins allows GDP to be deilivred to the nucleus where GEF converts GDP to GTP causing it to dissociate from NTF2 which then dissociates and brings in another Ran-GDP

Front 41

how might you confirm that the factor you have identified is necessary for promoting the nuclear uptake of Ran?

Back 41

The info in the problem states only that the cytoplasm passed over a Ran-GDP column is depleted of some factor that is essential. The inference is that NTF2 is the critical factor and so to prove that it is, you need to show that purified Ntf2 can promote the uptake of Ran-GDP into nuclei.

Front 42

How do you suppose that binding of methotrexate interferes with mitochondrial import?

Back 42

The binding of methrotrexate to the active site prevents the enzyme from unfolding, which is necessary for import into mitochondria.

Front 43

Why do mitochondria need a fancy transloactor to import proteins across the outer membrane? Their outer membranes already have large pores formed by porins

Back 43

These pores are large enough for all ions and metabolic intermediates but not large enough for most proteins

Front 44

Where is catalase located in cells without peroxisomes? Why does catalase show up as small dots of fluorescence in normal cells?

Back 44

it is located in the cytosol of peroxisome-deficient cells. It appears as small dots because its located in perozisomes which are small and distributed throughout cytosol

Front 45

What would you predict would be the effect of converting the first hydrophobic transmembrane segment to a hydrophillic segment?

Back 45

It would give rise to a protein with N-term in the cytosol but all other segments in their original location.

Front 46

How is symmetry generated in ER and asymmetry generated in plasma membrane?

Back 46

Symmetry of phospholipids in the 2 leaflets of ER membrane is generated by a phospholipd translocator, called scrambalase, that rapidly flips phospholipids of all types back and forth between monolayers of the bilayer. The plasma membrane contains a different kind of translocator which is specific for phospolipids containing free amino groups. These flippases remove these specific phospholipids from external and transfer them to internal and so it is asymmetric,

Front 47

List the organelles in animal cell that obtain their proteins via gated transport, trasnmembrane trsnport, and vesicular transport

Back 47

Gated- nucleus
Transmembrane- ER, mitochondria, peroxisomes
Vesicular- Golgi apparatus, endosomes, lysosomes

Front 48

Which type of protein synthesis, in cytosol or ER do you think is responsible for the majority of protein synthesis in a liver cell?

Back 48

One way to approach this problem is to compare the relative volumes of the
compartments that are served by cytosolic and ER protein synthesis. The compartments that depend on ER protein synthesis—the
ER, Golgi apparatus, endosomes, and lysosomes—account for less than 20%
of the cell volume. On this basis, then, one would conclude that cytosolic pro-
tein synthesis is responsible for the majority of cellular protein synthesis. In cells that do not secrete large amounts of protein, the majority of protein synthesis is likely to occur in the cytosol.

Front 49

If you think of a protein as a traveler, what kind of vehicle would best describe the sorting receptor?

Back 49

A taxi- anyone who has the money-the sorting signal-is taken on the journey

Front 50

If membrane proteins are integrated into the ER membrane by means of protein translocators, which are themselves membrane proteins, how do the 1st protein translocators become incorporated into the ER membrane?

Back 50

New cell membranes are made by expansion of exist-
ing membranes; the ER is never made from scratch. There will always be an
existing piece of ER with old translocators to integrate the new translocators.
Inheritance is therefore not limited to the propagation of the genome; a
cell’s organelles must also be passed from generation to generation. This is a chicken vs egg question

Front 51

T/F The nuclear membrane is freely permable to ions and other small molecules under 5000 daltons

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true because the membrane has many nuclear pores that allow small water soluble molecules to pass

Front 52

T/F Some proteins are kept out of the nucleus, until needed, by inactivating their nuclear localization signals by phosphorylation

Back 52

True. gene regulatory proteins are subject to this

Front 53

T/F all cytosolic proteins have nuclear export signals that allow them to be removed from the nucleus when it reassembles after mitosis

Back 53

false. Resident proteins of the cytosol do not have nuclear export signals

Front 54

How is it that a single nuclear pore complex can efficiently transport proteins that possess different kinds of nuclear localization signal?

Back 54

because transport is mediated by a variety of nuclear import receptors that are encoded by a family of related genes. Each gene product is specalized for transport of a group of proteins that share structurally similar localization signals. Yet, all the family membares share common features that allow them to interact iwht the nuclear pore complexes

Front 55

You have modified GFP to carry a nuclear localization signal at its N-term. How might the addition of an inhibitor of nuclear export to such cells resolve this issue.

Back 55

Proteins with a nuclear export signal also have a nuclear localization signal so these proteins typically shuttle between the nucleus and cytoplasm

Front 56

Why do you suppose it is critical that nuclear localization signals remain attached to their proteins?

Back 56

At each mitosis, the contents of the nucleus and cytosol mix after the nuclear envelope dissasembles. When the nucleus reassembles, the nuclear proteins must be selectively reimported. If the signals were removed, the proteins would be trapped in the cytosol after mitosis.

Front 57

The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins

Back 57

true

Front 58

the 2 signal sequences required for transport of nucleus encoded proteins into the mitochondrial inner membrane via the TIM23 complex are cleaved off the protein in different mitochondrial compartments

Back 58

False. Only one of the 2 is cleaved, the n-term

Front 59

import of proteins into mitochondria and chloroplasts is very similar, even the individual components of their transport machinery are homologous, as befits their common evolutionary origin

Back 59

false. components of the import machinery are not related

Front 60

Describe in a general way how you might use radiolabeled proteins and proteases to study import processes in osolated, intact mitochondria. What sorts of experimental controls might you include?

Back 60

Incubate the labeled proteins with isolated mitochondria under the conditions you wish to test, allow time for import, and then treat mixture with a protease. Proteins that arent imported will be digested by proteease.

Front 61

T/F the signal peptide binds to a hydrophobic site on the ribosome causing a pause in protein synthesis, which resumes when SRP binds to the signal peptide

Back 61

false

Front 62

T/F nascent polypeptide chains are transferred across the ER through a pore in the Sec61 protein translocator complex

Back 62

true

Front 63

T/F the Er lumen contains a mixture of thiol containing reducing agents that prevent the formation of S-S linkages by maintaining the cysteine side chains of th elumenal proteins

Back 63

false. ER lumen does not contain reducing agents

Front 64

Some membrane proteins are attached to the cytoplasmic surface of the plasma membrane through a C-terminal linkage to GPI anchor

Back 64

false. GPE anchored proteins are attached to teh external surface of plasma membranw

Front 65

explain how mRNA molecules can remain attached to the ER membrane while the individual ribosomes trasnslating it are released and rejoin the cytosolic pool of ribosomes

Back 65

An mRNA molecule is attached to ER by the ribosome translatting it. The ribosome population is not static and as mRNA moves through ribosomes, those ribosomes that are done traslating dissociate from the 3' end. But the mRNA remains attached to the ER by other ribosomes that are still in the process of translating it

Front 66

Why are cytosolic Hsp70 chaperone proteins required for import of proteins into mitochondria and chloroplasts but not for cotranslational import into ER

Back 66

Protein import into mito and chloro occurs as a post-translational event and chaperones are required to keep the newly made proteins in an unfolded conformation so that they can be imported. During co-translational import, the protein is being made and imported at the same time so there is no chance of it folding and no need for chaperones

Front 67

where would you expect to find microsomes in an electron micrograph of a liver cell

Back 67

nowhere. Microsomes are generated when cells are disrupted by homogenization

Front 68

Which one of the following might be expected to increase the effieciency of import if added to the mixtre of a protein and microsomes

Back 68

cytosolic Hsp70 would be expected to bind to the import protein and aid its unfolding, which will increase uptale.

Front 69

Compare and contrast protein import into the ER and into the nucleusMo

Back 69

Most proteins are translocated into ER as they are being made by ribosomes. In all cases, proteins are translocated across the membrane as unfolded polypeptide chains. Proteins import into nucleus after they have been synthesized, folded, and assembled into complexes. Signal sequences for ER import are often cleaved off after import, whereas nuclear localization signals are not.