Lecture 22: Protein Targeting

signal sequences act like a ________ for the proteins to be transported to the correct location

zip codes

signal sequences are NOT required for proteins destined for the ________

cytosol

as the protein is being made, the __ terminus of the growing polypeptide funnels out though an ________ in the ________ subunit

N; exit tunnel; large

the exit tunnel can hold a protein chain of about ________ amino acids

30

not much folding occurs inside the ________

exit tunnel/ribosome

the protein will start folding while it is ________

translated

once the peptide is finished being translated it is ________ and will finish ________

released; folding

a protein that is targeted to the ER will usually have a signal sequence located at the ________ or ________

N-terminal; internally

is the signal sequence of a protein bound for the ER usually removed?

depends

what is the signal sequence for proteins targeted for the ER? (generally)

hydrophobic sequence preceded by positively charged aa

proteins targeted for the mitochondrial matrix will usually have ________ signal sequences located at the ________

multiple; N-terminus

why do mitochondrial matrix proteins need multiple signal sequences?

multiple membranes to pass through

are signal sequences removed from proteins bound for the mitochondrial matrix?

yes

what is the signal sequence of a protein targeted for the mitochondrial matrix?

amphipathic helix with R/K and hydrophobic sides

where is the signal sequence located for proteins targeted to the peroxisome?

C terminal

are the signal sequences pf proteins bound for peroxisomes usually removed?

no

what is the signal sequence for proteins bound for the peroxisome?

SKL at C-term

for proteins targeted to the nucleus, where are their signal sequence usually located?

internally

are signal sequences for proteins targeted to the nucleus usually removed?

no

what is the signal sequence for proteins bound for the nucleus?

clusters of basic aa separated by ~10aa

________ direct proteins to the nucleus

nuclear localization signals (NLSs)

the NLS is located ________ and is ________ cleaved off

internally; NOT

what are two ways to see experimentally determine is a sequence is actually a sign sequence?

mutate sequence (still targeted?); add to another protein (targeted to same place?)

proteins targeted for the ER are often part of the ________ pathway

secretory

membrane targeting requires:

cotranslational protein translocation

once ribosomes start translation, they can be moved to the ________ to finished translation

ER

what is the name of the protein where ribosomes doc at the ER?

preprotein translocase

Preprotein translocase is called the ________ complex

Sec61

Sec61 is a protein-________ channel with an ________ pore that spans the ER membrane

conducting; aqueous

what is the main function of Sec61?

pass protein across the ER membrane

Sec61 proteins have a ________ that can block the protein from passing through

plug

how does preprotein translocase get into the membrane?

via another preprotein translocase

where does the Sec61 protein bind to the ribosome?

at the exit site

N-terminal hydrophobic signal sequences direct secreted and membrane proteins to the ________ (protein)

translocase

if the N-terminal has the ER signal sequence, translation will be ________, and the ribosome will be moved to the ________ where it will ________ translation

stopped; ER membrane; resume

are signal sequences the same for all proteins?

no; its more structure than specific aa

the signal sequence will be recognized by a ________ (protein)

signal recognition particle

what does SRP stand for?

signal recognition particle

what are the three functions of the SRP?

recognize signal sequence; pause translation; direct to ER membrane

the SRP is a ________

ribonucleoprotein

the SRP is made up of ________ proteins and ________ RNA molecule/s

5; 1

where does the SRP molecule bind to the ribosome?

sits over A site, prevents another tRNA

SRP has a signal sequence binding ________, which will recognize the ________ regions of the signal sequence

pocket; hydrophobic

the SRP reaches from the ________ across the ribosome to the ________

exit tunnel; A site

once SRP paused translation, it will bind to the ________

SRP receptor

once the SRP binds to the SRP receptor, it will undergo ________, allowing the ribosome to bind fully to the ER channel

GTP hydrolysis

both SRP and SR are bound to ________, but neither are able to hydrolyze it by themselves

GTP

GTP hydrolysis only occurs when ________ bind to ________ (ER signaling pathway)

SRP; SRP receptor

the SR receptor is associated with the ________,while the SRP is associated with the ________

translocon; ribosome

GTP hydrolysis lets the SRP know that it has arrived at the ________ and it can allow ________

translocon; translation to reoccur

The region around the ribosome exit tunnel so conserved because it has to interact with both the ________ AND the ________

SRP; pre protein translocase

what protein cleaves off the signal sequence?

signal peptidase

once the protein translator is activated, the polypeptide is ________ through the channel into the ________

pulled; ER

what proteins pull the polypeptide through the translocon?

chaperones

as the polypeptide is being threaded through, the ________ of the sequence is held onto

hydrophobic (signal sequence)

once the polypeptide is fully threaded through the membrane; the signal sequence is ________, releasing a ________ into the ER lumen

cleaved off; mature protein

where does the signal sequence go once it is cleaved off?

stays in the membrane

when an integral membrane protein at signaled to the ER; it has a ________ and a ________ sequences

start transfer; stop transfer

the region of an integral membrane that sits in the membrane is ________

hydrophobic

when an integral protein is being threaded through the translocon; a ________ region will cause threading to stop, and induce the ________ of the signal sequence

2nd hydrophobic; cleavage

in an integral membrane protein (in the ER); the ____ terminal will be in the inside of the ER membrane, while the ____ terminal will be in the cytoplasm

N; C

what are polytonic membrane proteins?

have multiple intermembrane domains(threaded in and out several times)

polytonic membrane proteins need multiple ________

start/stop sequences

how does the polytonic membrane protein know how many times to pass through the membrane?

multiple start/stop regions

________ charges on the polytonic membrane like to be on the inside of the ER, which helps determine which ________ will be on the inside vs outside of the membrane

positive; portions (regions)

pre protein translocase allows for ________ escape of signal peptides and transmembrane segments into the ________

lateral; membrane

proteins are glycosylated in the ________ and ________

ER; Golgi

glycosylation is important for ________, ________, and ________

folding; function; targeting

what are the two types of glycosylation?

N-linked and O-linked

most proteins in the secretory pathway are ________

glycosylated

glycosylation is the process of adding ________ to protein

sugar

N-linked glycosylation occurs in the:

ER and Golgi

what is the N-glycosylation signal? (specific amino acids)

Asn-A-Ser/Thr

glycosylation occurs at ________ amino acid sequence

specific

in N-linked glycosylation, the sugar is attached to the Asn ________

side chain

what is a sugar attached to an amino acid called?

amino sugar

amino sugars have a/an ________ group instead on a/an ________ group on their first carbon

amino; hydroxyl

N-linked oligosaccharides are added by ________ (mechanism) in the ER

block transfer

when does N-linked glycosylation occur?

cotranslationally!

is glycosylation templated?

no

in N-linked glycosylation; there is a ________ transfer of sugars, and then they are ________ down to core __ sugar residue

large; trimmed; 5

after the sugars are trimmed down to the few main ones, sugars can be ________ in varying combinations

added

sugars are added from ________ sugar donors

nucleotide

sugars are ________ before glycosylation which provides the energy needed to form the ________ bond

activated; glycosidic

in the activation of sugar for glycosylation; the nucleotide is added to the ________ carbon

1’

once the sugar is added to the amino acid, ________ is released

NDP

O-linked glycosylation occurs in the ________

Golgi

most glycosidic bonds require a specific ________

glycosyltransferase

O-linked glycosylation occurs at ________ or ________ residues

ser; thr

in o-linked glycosylation; the chains of sugars are build up ________

one at a time

O-linked glycosylation occurs on the ________ group of serine or threonine

OH

N-linked glycosylation occurs on the ________ group of Asn

NH2

O-linked glycosylation does not require a specific ________ motif

sequence (unlike N-linked)

does O-linked glycosylation occur cotranslationally?

no; it is done on completed polypeptide chains

O-linked glycosylation occurs by the ________ addition of ________ monosaccharide units

serial; single

N-linked glycosylation occurs by the ________ transfer of a ___-residue core oligosaccharide; which is later ________. However it will ALWAYS retain the same___-residue core polysaccharide

block; 14; modified; 5

the addition of a sugar occurs at the ________ of the amino acid, not the ________

side chain; backbone

because glysosylation is not template driven, it tends to be ________, forming many different ________ of the same protein

heterogeneous; glycoforms

what are the 5 main functions of glycosylation?

protein folding/structure, recognition, targeting, antigens, hiding antigens

I- cell disease is caused by the inability to ________

target proteins to the lysosome