Biol 266 Unit 6-1

Location of Protein Na+/K+ ATPase

Plasma membrane

Location of RNA polymerase

Nucleus

Location of Proteases

Lysosome

Location of catalase

Peroxisome

Location of ATP synthase

Mitochondria

Location of Hormones

Extracellular Space

Most of the proteins synthesized in eukariotic cells are encoded by __________.

nuclear DNA

Most of the proteins synthesized in eukariotic cells are synthesized on ___________ in the cytosol.

Ribosomes

Most of the proteins synthesized in eukariotic cells are delivered to the organelle of destination from the ____________.

cytosol

A few of the proteins synthesized in eukariotic cells encoded by the DNA present in ___________ and chloroplast .

mitochondria

A few of the proteins synthesized in eukariotic cells synthesized on _________ inside mitochondria and chloroplasts.

ribosomes

A few of the proteins synthesized in eukariotic cells are incorporated directly into compartments within mitochondria and ___________.

chloroplasts

Protein sorting:

The process by which newly-made proteins are directed to the correct location.

Retention in lumen of ER, Signal Sequence:

-Lys-Asp-Glu-Leu-COO—

Import into nucleus, Signal Sequence:

-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-

Import into Peroxisomes, Signal Sequence:

-Ser-Lys-Leu-COO—-

Signal sequences are __________ and ___________ for protein sorting

necessary ; sufficient

3 steps in protein sorting:

Recognition of signal sequence / Targeting to the outer surface / Import or transport of the protein.

Protein sorting: ___________ of signal sequence by a shuttling cytosolic ____________/ __________ to the __________ surface of the organelle membrane / ________ of the targeted protein into the membrane or ___________ of the protein across the membrane.

Recognition ; receptor ; Targeting ; outer ; Import ; transport

Three main mechanisms to import proteins into a membrane-enclosed organelle:

Transport through nuclear pores / Transport across membrane / Transport by vesicles.

Transport through nuclear pores, Transports specific

_______ that remain ______ during transport.

proteins ; folded

Transport across membranes

ER, mitochondria, chloroplasts, peroxisomes. Requires protein __________. Proteins are ___________ in order to cross the membrane.

translocators ; unfolded

Transport by vesicles

From ER onward and through __________ system. Transport vesicles collect cargo protein and ________ from membrane. Deliver cargo by _______ with another compartment. Proteins remain _______ during transport

endomembrane ; pinch off ; fusing ; folded

Entry into the nucleus proceeds through a protein structure called the _________________________.

nuclear pore complex (NPC)

NPC Can transport _______ molecules/sec, both directions _______________ .

1000 ; simultaneously

Small, water-soluble molecules and proteins up to ~40 kDa can move through the nuclear pore complex (NPC) by_________________

passive diffusion.

When an unfolded protein goes back, it will go through the __________of the ER and interect with _________.

quality control ; Calnexin

The Ran “gradient” ensures ___________ to nuclear transport.

directionality

The GTP-bound form only exists in the ________.

nucleus

The GDP-bound form only exists in the _________. “

cytosol

For import into the matrix, a (usually) ________________ is required. If the protein localizes to the intermembrane space, a __________ sorting sequence is needed.

N-terminal sorting sequence ; second

Matrix-targeting sequences are rich in _________, _________-charged and hydroxylated (Ser, Thr) residues, but lack __________residues. Tend to form ___________ helix.

hydrophobic ; positively ; acidic ; amphipathic

Import into the mitochondria only occurs at points where the inner and outer membranes are in __________.

close contact

Mitochondria import : 1. Precursor proteins are kept in an unfolded state by the action of the cytosolic chaperone _______. This requires ________ in the form of ATP hydrolysis.

Hsc70 ; energy

Mitochondria import : 2. The matrix-targeting sequence then interacts with a _________ in the outer mitochondrial membrane called ______ or ______.

receptor ; TOM20 ; TOM22

Mitochondria import : 3. The receptor transfers the protein to the ______________ of the _______ membrane composed of the protein ________.

general import pore ; outer ; TOM40

Mitochondria import : 4,5. At contact sites between the inner and outer membranes, the protein passes through the ________ of the _______ membrane composed of the proteins _____ and _____.

import pore ; inner ; TIM23 ; TIM17

The proteins ___ are in the intermembrane, while _______ is in the outer membrane.

TIM ; TOM

_____ comes before ______.

TOM ; TIM

Mitochondria import : 5. _____ _____ binds to _____. _________ by this complex helps power translocation of the protein into the matrix

Matrix Hsc70 ; TIM44 ; ATP hydrolysis

Mitochondria import : 6. As the matrix-targeting sequence emerges in the matrix, it is cleaved by a matrix ________.

protease

Mitochondria import : 7. The protein can then _____ into its final conformation, often (but not always) assisted by matrix __________.

fold ; chaperonins

The H+ electrochemical gradient generated by _________________ is also required for protein import into mitochondria. This ensures that only _________that are actively respiring can import proteins. Hence, ____________ block import.

oxidative phosphorylation ; mitochondria ; uncouplers

ER import : most of the proteins that enter the endoplasmic reticulum begin to be ___________ across the ER membrane before the protein is completely _____________.

translocated ; synthesized

Two separate populations of ribosomes in the cytosol:

Membrane-bound ribosomes and free ribosomes.

Membrane-bound ribosomes are attached to the _______________ of the ER membrane and are ____________ proteins that are translocated into the ER.

cytosolic surface ; synthesizing

Free ribosomes are ____________ to any membrane and are _____________ all of the other proteins.

unattached ; synthesizing

Importing a soluble protein into the ER lumen : 1,2. The emerging polypeptide with its ER signal sequence exposed is engaged by a complex of six _________ and an associated RNA molecule called the ____________________. This binding halts ________ and delivers the ribosome/polypeptide to the ER.

proteins ; signal recognition particle (SRP) ; translation

Importing a soluble protein into the ER lumen : 3. SRP delivers the ribosome/polypeptide to the ________________. This interaction is enhanced by the __________ to both SRP and its receptor.

SRP receptor ; binding of GTP

Importing a soluble protein into the ER lumen : 4. The ribosome/polypeptide is then transferred to the ___________, inducing it to open and receive the polypeptide which enters as a loop. _____________ by SRP and its receptor free these factors for another round of import.

translocon ; Hydrolysis of GTP

Importing a soluble protein into the ER lumen : 5,6. Translation then resumes and the signal sequence is _________ by a membrane-bound protease called ______________. Following this digestion, the rest of the protein is _____________ and enters the lumen of the ER.

cleaved ; signal peptidase ; synthesized

Importing a soluble protein into the ER lumen : 7,8. Following completion of ________ , the ribosome is __________ causing the ___________to close. The newly-synthesized protein then _______.

translation ; released ; translocon ; folds

6 main types of membrane-anchored proteins:

Type I, Type II, Type III, Tail-anchored, Type IV and GPI-anchored.

Type 1 : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(Yes/no).

Yes ; Yes

Type 2 : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(Yes/no).

No ; Yes

Type 3 : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(Yes/no)

No ; Yes.

Tail-anchored : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(Yes/no).

No ; No

Type 4 : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(YES/NO).

No ; YES

GPI-anchored : Cleavable N-terminal signal sequence ________ (Yes/No). SRP/SRP receptor ________(Yes/no).

Yes ; Yes

Type 1 : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (In/Out).

Single Pass ; Out ; In

Type 2 : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (In/Out).

Single Pass ; In ; Out

Type 3 : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (IN/OUT).

Single Pass ; OUT ; IN

Tail-achored : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (IN/OUT).

Single Pass ; IN ; OUT

Type IV — A : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (In/Out).

Multispanning ; In ; In

Type IV — B : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (In/Out).

Multispanning ; Out ; In

GPI-anchored : _________(single pass / Multispanning / Entirely lumal [out]). N____ — C_____ (In/Out).

Entire protein is lumenal (out) ; Does not Apply

Type I membrane proteins use a cleavable signal sequence and a _______________ sequence that acts as the membrane spanning domain. The ________ opens to ________ this hydrophobic stretch into the membrane.

stop-transfer anchor (STA) ; translocon ; release

Type II and _____membrane proteins use a _______________ sequence that acts as a dual signal sequence (directing the protein to the ER by the SRP) and the anchor or membrane-spanning sequence.

III ; signal-anchor (SA)

In Type II and III membrane proteins, the orientation is determined by the positioning of the _________ within the translocon. This is in turn determined by the positioning of ____________ residues relative to the SA sequence. Those residues remain ________.

SA sequence ; positively-charged ; Cytosolic

If the positively-charged residues are between the N-terminus and the SA, then it will be________.

Type II

If the positively-charged residues are between SA and C-terminus, then it will be ___________.

Type III

Tail-anchored proteins are inserted into the ER after ___________ is ___________ since the hydrophobic stretch that is inserted into the bilayer needs to fully emerge from the ribosome.

translation ; completed

The _________ orient Type 2/3 proteins so that the positive residue are on the _________side.

translocon ; cytosolic

Diff between SA and STA : SA will be ___________ by ____ . STA is being transported into the __________ which _____ the translocon. (____does not meet the SRP) (not recognized SRP but recognized by translocon)

recognized ; SRP ; translocon ; stops ; STA

Tail-anchored proteins : 1. The hydrophobic tail binds to ____ in its _______ state. Three other proteins (Get4/5 and Sgt2) bind the hydrophobic tail first before transfer to Get3.

Get3 ; ATP-bound

Tail-anchored proteins : 2. The Get3-polypeptide complex then binds to the ___________ on the ER membrane.

Get1/2 complex

Tail-anchored proteins : 3. Get3 then ______________ and releases the polypeptide, embedding its hydrophobic stretch into the ER membrane.

hydrolyzes ATP

Tail-anchored proteins : 4. Get3 binds ______ and is released from the membrane, ready for another round of activity.

ATP

GPI (glycosylphosphatidylinositol)-anchored proteins insert into the ER like Type I membrane protein using a __________________________ sequence. An enzyme (___________) then (i) __________ the protein within the lumen of the ER and (ii) _________it to the assembled GPI anchor.

stop-transfer anchor (STA) ; transamidase ; cleaves ; transfers

The purpose of transferring one lipid anchor for another:

1. The GPI anchor more readily _________ in the membrane

2. GPI can act as a __________ signal.

diffuses ; targeting

Type IV membrane proteins use combinations of ____________________ and __________________ sequences.

stop-transfer anchor (STA) ; signal-anchor (SA)

If the first SA sequence is a Type II SA, then the protein will be N_____.

IN

If the first SA sequence is a Type III SA, then the protein will be N______.

OUT

The more ________________ an amino acid is, the more positive the hydropathic index.

hydrophobic

The more hydrophilic the amino acid, the more ___________ the hydropathic index.

negative

In the hydropathic Index, Type 1 has hydrophobic __________________ in the beginning of the protein and a _______________________in the middle.

cleavable signal sequence ; Stop Transfer Sequence

In the hydropathic Index, Type 4 has more than 1 ______________. No hydrophobic peak at the _________. 

membrane spanning domain ; beginning

In the hydropathic Index, Type 2 or Type 3 do not have cleavable signal sequence but they have a _____________.

signal anchor sequence

In the hydropathic Index, If positive amino are on the left, this means the amino terminus is __________ and its ________.

outside ; Type 2

In the hydropathic Index, If positive amino are on the right, the amino terminus is going to be in the _______ (Looks like _____) and its ______.

luman ; Type 1 ; type 3

In the hydropathic Index, In Tail anchor protein you would not see any _________, you would only see one hydrophobic peak at the very _____.

peaks ; end

In the hydropathic Index, In GPI anchor protein , we have one signal sequence in the _________ and one in the _________.

beginning ; end

The ER is the starting point for:

1. Soluble proteins that will be ____________ from the cell.

2. _____________ that are destined for the Golgi, lysosome or endosomes.

3._______________ that will embed in the Golgi, lysosome, endosomes or plasma membrane.

secreted ; Soluble proteins ; Membrane proteins

Four principle modifications that occur in the ER:

Disulfide bond formation / Glycosylation / Folding of polypeptides chains and assembly of multisubunit complexes / Proteolytic cleavage of amino-terminal signal sequences

Disulfide bond formation –____________ formation between thiol groups of cysteine residues either on the same protein (_____________) or on two different proteins (_______________)

covalent bond ; intramolecular ; intermolecular

Disulfide bond formation is dependent upon the ER resident enzyme protein disulfide isomerase (_______). Thus, only (i) ____________ or (ii) lumenal or extracellular domains of ____________ undergo this modification.

PDI ; secreted proteins ; membrane proteins

Disulfide bonds______________ – important for proteins that will be subjected to either extremes in pH or environments with high levels of proteases.

stabilize protein structure

Glycosylation – begins with the addition of a common ____________ addition to __________ residues in the consensus sequence_______________ .

oligosaccharide ; asparagine ; Asn-X-Ser/Thr

Referred to as _____________ since the oligosaccharide is added to the amine group of asparagine.

N-linked glycosylation

In glycosylation, the precursor ________________ is transferred to the protein as the consensus sequence emerges from the ____________. Requires an ER membrane – bound enzyme complex called _________________.

oligosaccharide ; translocon ; oligosaccharyl transferase