Mol Cell - Exam 3

nuclear envelope

separates the nuclear contents from the cytoplasm

what does the nuclear envelope control

traffic of proteins and RNAs through nuclear pore complexes, and plays a critical role in regulating gene expression

what does the nuclear envelope consist of

two nuclear membranes, an underlying nuclear lamina, nuclear pore complexes

outer membrane is

continuous with the endoplasmic reticulum

inner membrane has

proteins that bind the nuclear lamina

nuclear membranes are

phospholipid bilayers permeable only to small nonpolar molecules

nuclear pore complexes

are the only channels for small polar molecules, ions, and macromolecules

nuclear lamina

a fibrous mesh that provides structural support, that consists of fibrous proteins (lamins) and other proteins

lamins

intermediate filament proteins that associate to form higher order structures

what happens when two lamins interact to form a dimer

the α helical regions wind around each other to form a coiled coil

what do mutations in lamin genes cause

several inherited tissue-specific diseases, the bases of the pathologies in each of these diseases is still unclear

what do lamins bind to

inner membrane proteins such as emerin and lamin B receptor (LBR), connected to the cytoskeleton by LINC protein complexes, they also bind to chromatin

nuclear pore complexes

composed of about 30 pore proteins (nucleoporins)

what must happen to RNA before being exported

rna synthesized in the nucleus must be exported to the cytoplasm for protein synthesis

molecules pass through pore complexes by what mechanisms

small molecules and protein pass freely in either direction, proteins and rnas are selectively transported; recognized by specific signals

nuclear localization signals

proteins that must enter the nucleus have amino acid sequences

nuclear transport receptors

where nuclear localization signals are recognized, the amino acid sequence responsible for nuclear localization using T antigen mutants, when the same sequence was attached to other proteins, they are transported to the nucleus

what must happen to proteins from synthesis sites?

proteins needed for nuclear functions must be imported from synthesis sites in the cytoplasm

T antigen nuclear localization signal

single stretch of amino acids in rich basic amino acid residues (lysine and arginine)

what is the Lys-Arg sequence

Lys-Arg, followed by a Lys-Lys-Lys-Lys sequence located ten amino acids farther downstream

what do NLS recognize

receptors called importins, which carry proteins through the nuclear pore complex

what do importins work in

conjunction with the GTP-binding protein RAN, which controls direction of movement

what do importins bind to

the NLS of a protein, then to nuclear pore proteins and the complex is transported across the membrane

what do Ran/GTP bind to

importin and this complex is transported back, Ran GAP hydrolyzes the GTP on Ran to GDP, releasing importin

where is Ran/GDP formed

in the cytoplasm, transported back to the nucleus by its own import receptor, where Ran/GTP is regenerated

where are proteins exported

from the nucleus

what do proteins target

for export by amino acid sequences called nuclear export signals (NES)

what recognizes NES

receptors in the nucleus (exportins), which direct transport to the cytoplasm

what are importins and exportins known as

members of a family of nuclear transport receptors (karyopherins)

what do exportins form

stable complexes with cargo proteins in association with Ran/GTP in the nucleus

in the cytoplasm, GTP hydrolysis does what

release of Ran/GDP leads to dissociation of the cargo protein

what do exportins karyopherin transport

tRNA, rRNA, miRNA - function in cytoplasm

what is the function of snRNA in the nucleus

pre-mRNA splicing

what is the function of snoRNA

rRNA processing

what does mRNA transport not involve

karyopherins and its independent of Ran

what does a distinct transporter complex do

moves the mRNA through the nuclear pore

helicase

on the cytoplasm side releases the mRNA and ensures unidirectional transport

small nuclear ribonucleoprotein particles (snRNPs)

small nuclear RNAs (snRNPs), are complexed with 6-10 protein molecules to form a snRNPs

regulation of protein transport

is a mechanism for controlling protein activity in the nucleus

EX : regulation of import and export of transcription factors is a way of controlling gene expression

what do transcription factors or other proteins associate with

cytoplasmic proteins that mask their NLS, remaining in the cytoplasm

what is transcription factor NF-kb complexed with

IkB in the cytoplasm

• if IkB is phosphorylated and degraded by ubiquitin-mediated proteolysis NF-kB can enter the nucleus and activate transcription of its target genes

what happens to chomatin during mitosis

becomes highly condensed to form the compact metaphase chromosomes

interphase

most chromatin decondenses and is distributed throughout the nucleus, chromosomes occupy distinct regions and are orgnanized such that transcriptional activity is correlated with its position

DNA replication an transcription takes place in clustered regions

what does each chromosome occupy

a discrete region of the nucleus - a chromosome territory

euchromatin

interphase cells, that are decondensed and transcriptionally-active, and distributed throughout the nucleus

heterochromatin

highly condensed and not transcribed, often associated with the nuclear envelope or periphery of the nucleolus

dna replication

replication factories

where does transcription occur

at clustered sites (transcription factories)

nuclear bodies

organelles within the nucleus that concentrate proteins and RNAs that function in specific nuclear process

nucleolus funcitons

in rRNA synthesis and ribosome production

5.8S, 18S, and 28S rRNA

transcription in nucleolus by RNA polymerase I; 45s ribsomal precursor RNA

transcription of the 5S rRNA

outside the nucleolus; RNA polymerase III

nucleolar organizing regions

nucleolus organized around the chromosomal regions that contain the 5.8S, 18S, and 28S rRNA genes

three regions nucleoli consist of

fibrillar center, dense fibrillar component, and granular component

FC

genes encoding rRNA

interface of FC and DFC

transcription

DFC

pre-rna is processed

G

assembly of ribsomal subunits

what does the processing of pre-rRNA require

action of both protein and RNAs that are localized to the nucleus

ribosome formation

pre-rRNA, ribosomal proteins, 5S rRNA

where are ribosomal proteins produced

in the cytoplasm and imported to the nucleolus

5S rRNA

are transcribed outside of nucleolus and produced elsewhere in the nucleus, assemble to form pre-ribsomal particles

pre-ribosomal particles

are exported to the cytoplasm, yielding the 40S and 60S ribsomal subunits

what do the ER, golgi, endosomes, and lysosomes do?

protein process and connected by vesicular transport

endoplasmic reticulum (ER)

network of membrane-enclosed tubules and sacs - that extends from nuclear membrane throughout cytoplasm

rough ER

ribosomes on the outer surface

smooth ER

lipid metabolism

what are newly sythesized proteins labeled with

radioisotopes

secretory pathway

rough ER, golgi, secretory vesicles, cell exterior

proteins synthesized on free ribosomes

stay in the cytosol or are transported to the nucleus and other organelles

proteins sythesized on membrane-bound ribsomes

are translocated directly into the ER through translocon

cotranslational translocation

proteins move into the ER during their synthesis on membrane bound ribosomes

posttranslational translocation

proteins move into the ER after translation has been completed on free ribosomes

where does protein synthesis start

on ribosomes that are free in the cytosol

contranslational pathway

ribosomes are targeted to the ER by a signal sequence at the amino terminus which is removed when the growing polypeptide chain enters the ER

postranslational translocation

proteins move into the ER after translation has been complete on free ribosomes, starts on ribosomes that are free in the cytosol

vesicles derived from

ER were added, the signal sequences were removed by proteolytic cleavage

what do signal sequences include

a stretch of hydrophobic residues, and usually located at the amino terminus of the polypeptide chain

what is the first step of contranslational targeting

step 1; as the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP)

what is the second step of contranslational targeting

the SRP is released, the ribosome binds to the translocon, and insertion of the signal sequence opens the translocon

what is the third step of contranslational targeting

the SRP is released, the ribosome binds to the translocon, and insertion of the signal sequence opens the translocon

what is the fourth step of contranslational targeting

translation resumes and the signal sequence is cleaved by signal peptidasee

what is the fifth step of contranslational targeting

continued translation drives translocation of the growing polypeptide chain across the membrane

what is the sixth step of contranslational targeting

the completed polypeptide chain is released within the ER lumen

lumen of the ER

equivalent to the exterior of the cell

amino terminal signal sequence

cleaved by signal peptidase during translocation through translocon

transmembrane α helix

middle of the protein that halts translocation and anchors the polypeptide in the membrane

internal transmembrane sequence

inserted directly into the ER membrane, recognized by SRP, but not cleaved by signal peptidase

transmembrane α helices

exit the translocon laterally and anchor proteins in the ER membrane

protein folding and processing can occur

during translocation across the ER membrane or within the ER lumen

primary role of lumenal ER protein is to

assist and fold, assembling newly translocated polypeptides

reducing environment

most cysterine residues are in their reduced state

oxidizing environment

promotes disulfide (S-S) bond formation, facilitated by protein disulfide isomerase

asparagine residues

proteins are glycosylated on (N-linked) as they are translocated into the ER

oligosaccharide

is sythesized on a lipid (dolichol) carrier

what does glycosylation help prevent

protein aggregation in the ER and provides signals for subsquent sorting

glycosylphosphatidylinositol (GPI) anchors

attached to the plasma membrane by glycolipids, that are assembled in the ER membrane and added to the carboxy terminus of some polypeptides

misfolded proteins

removed from the ER by ER associated degradation

• identified, returned to cytosol, degraded by the ubiquitin-proteasome system

• chaperones and protein processing enzymes in ER lumen: sensors of misfolded proteins

as glycoprotein exits the tranlocon, chaperones bind and assist in folding

if protein is correctly folded it exits the ER, however if too many hydrophobic regions are exposed, indicating improper holding, the protein is targeted back to the cytosol through a ubiquitin ligase complex in the ER membrane

unfolded protein response (UPR)

if an excess of unfolded proteins accumulates, a signaling pathway is activated