Cellular Biology Exam #2

Nuclear Envelope

Two membranes: inner & outer.
Outer is continuous with ER.

Lamin

Provide structure support to nucleus.
Intermediate filament.
Globular head and tail, central rod domain.

Hutchinson Gilford progeria syndrome

Very rare.
Mutation in lamin A gene that leads to premature aging because lamin A gene is important for nucleus envelope stability.

Nuclear Lamina

Connected to inner membrane via LBR & emerin.
Connectged to cytoskeleton via KASH & SUN.

Emerin & LaminB receptor (LBR)

Integral proteins that bind lamins to the inner membrane of the nucleus.

SUN & KASH

Integral membrane proteins that link the nuclear lamina to the cytoskeleton.

Import/export of nucleus

Small stuff (nucleotides) diffuse freely across the envelope.
Big stuff must be actively transported across.

Nuclear Pore Complex

How big stuff is actively transported across the nuclear membrane. Proteins are synthesized in the cytosol --> nuclear localization signals are included in the protein sequence --> peptide sequences function as zip codes for proteins.

Nuclear Localization Signals

Two kinds:
Linear Signal
Bipartite Signal

Which of the following does not take place in the nucleus?

Translation
- Takes place in the ribosome.

The outer nuclear membrane is contiguous with the ...

Endoplasmic reticulum.

The fibrous proteins underlying the inner nuclear membrane are ...

Lamins

Protein transport into the nucleus occurs by...

Selective transport through the nuclear pores.

"Classical" nuclear localization signals are composed primarily of ...

Basic amino acids.

The directionality of nuclear protein import is determined by ..

An unequal distribution of Ran/GTP.

The localization of Ran GAP in association with the cytoplasmic filaments of the nuclear pore results in the conversion of the nucleotide bound to Ran in the nucleus to ...

GDP.

An important step in the import of the transcription factor NF-kB into the nucleus is regulated by a ...

Protease.

mRNA molecules are exported from the nucleus to the cytoplasm via...

A recruited protein complex.

Chromosomes are distributed in the nucleus...

In distinct territories.

The most prominent nuclear body is the ...

Nuceleolus.

Which of the following are not subcompartments of the nucleus?

Lipid droplets.

The nucleolus is the site where ...

Ribosomal RNA is transcribed and ribosomes are partially assembled.

The nuclear pore complex of proteins is estimated to have a mass ...

About 30 times that of a ribosome.

The nuclear basket protein complex is a component of the nuclear pore complex found ...

Inside the nucleus.

Gene expression is one possible explanation for the phenotypic consequences of nuclear lamina diseases. The other is ...

Mechanical stress.

What is the average number of replication forks per DNA replication cluster in a mammalian cell nucleus?

Approximately 20

Most snoRNAs function as ...

Guide RNAs.

Which of the following factors contributes to the comparative ease of determining how pre-rRNA is processed?

The abundance of rRNA genes
The abundance of ribosomes (5-10 million) that need to be synthesized per cell cycle
The grouping of rRNA genes into discrete nuclear subcompartments, the nucleoli
All of the above

Which of the following statements about heterochromatin is false?

There are two forms—constitutive and facultative heterochromatin.

What is the function of karyopherins?

They transport macromolecules into or out of the nucleus.

Initial steps in the functional assembly of snRNPs occur in the ...

Cytoplasm.

Protein transport into the nucleus occurs by ...

selective transport through individual subsets of nuclear pore complexes that are specific for individual classes of proteins.

Ribosomes contain one copy each of 5.8S, 18S, and 28S rRNA. The major mechanism ensuring that each is produced in equal molar amounts is ...

the existence of common sequence features for transcription initiation for the genes encoding each rRNA.

Which of the following is a possible intermediate during pre-rRNA processing?

An RNA molecule containing 18S + 5.8S rRNAs

RAN

a small G-protein that is regulated by GTP binding.

GEF

Guanine exchange factors that switch GDP for GTP on the nuclear side of the envelope.

Ran GAP

makes Ran hydrolyze GTP to GDP on the cytosolic side.

Ran-GDP

CYTOSOL

Ran-GTP

NUCLEUS

Imporin

Nuclear import.

Exportin

Nuclear export.

Nuclear proteins

Made in cytosol --> nuclear destination. DOES NOT GO THROUGH ER.

mRNA export

RNA helicase remodels mRNA and prevents re-import.
mRNA binding proteins transported back to nucleus.

snRNA

small RNA that functions in nucleus - splicing of introns to make mature RNA.
snRNA made in nucleus --> out to cytosol to pick up protein to make nRNP --> back into nucleus.

Lamins

Intermediate filaments of the nucleus.
Docking site for chromatin & other proteins into functional regions (in animals).

2 types of chromatin

Heterochromatin: not actively transcribed, highly condensed, basically silent.

Euchromatin: actively transcribed, can make its own proteins.

Structures in Nucleus

Canal bodies - snRNP assembly
PML - transcriptional regulation, DNA repair.

Posttranscriptional mRNA processing

The addition of a 5' cap and a poly-A tail
Introns are removed by splicing.

RNA processing

1. transcription
2. 5' cap added
3. 3' cleavage
4. poly-A tail created
5. splicing begins
6. introns removed

Spliceosome

Pre-mRNA introns are spliced out by these.

Small nuclear RNAs

U1, U2, U4, U5, U6

Nucleolus

where some rRNAs are transcribed, processed, and assembled into ribosomes.

Ribosomal RNA gene structure

Multiple copies.
3 of the 4 ribosomal RNAs are transcribed as a single 45S pre-rRNA
The 45S pre-rRNA is then processed into 18S, 5.8S, and 28S rRNA.

How does rRNA processing occur?

By the action of snoRNA + proteins.
Directing the base modifications of pre-rRNA.
Example methylation

snoRNA

small nucleolar RNA

Assembly of pre-ribosomal particles and how they get out of the nucleus

Synthesized in the nucleus - 40S subunit (18S) and 60 subunit (28S, 5.8S)

Not made in the nucleus = 5S

Proteins Sorting - delivery from cytosol

DO NOT GO THROUGH ER
chloroplasts, mitochondria, interior of peroxisomes, interior of nucleus, cytosol, ER

Proteins Sorting - indirect delivery from ER in vesicles

Secreted proteins - GO THROUGH ER
ER, golgi, lysosomes, plasma membranes, nuclear membranes, peroxisomal membranes

Proteins sorting

synthesis of most proteins begins on ribosomes in the cytosol. all guided by sorting signals in the amino acid sequence. all require energy input.

Signal sequences

Direct proteins to the correct compartment. Signal sequences are necessary & sufficient to direct a protein to a particular organelle.

Endoplasmic Reticulum

Transitional ER: vesicles bud off golgi
Rough ER: associated with ribosome involved in protein synthesis.
Smooth ER: not associated with ribosome involved with lipid synthesis.

Rough ER

associated with ribosome.
involved in protein synthesis.
RIBOSOMES ATTACHED to the cytosolic surface of the ER.
specific site of protein synthesis - transmembrane proteins, secreted proteins, lysosomal proteins, & golgi proteins.

Smooth ER

not associated with ribosome.
involved in lipid synthesis.

How are proteins processed through the ER and golgi?

Classic pulse chase experiment
Pancreactic aclnar cells

learned that secretory proteins made in ER --> golgi --> vesicles --> outside of cell.

Cotranslational targeting of secretory proteins to ER.

SRP: signal recognition particle.

Function together in folding proteins

Chaperonin and Hsp70

Protein modification in the ER

N-linked sugars
S-S bridges formed by protein disulfide isomerase (PDI)
Addition of a GPI anchor to the C-terminus of the protein

Lipid modification of proteins

Added in cytosol (all ylations): N-myristoylation, prenylation, palmitoylation

Added in ER: GPI anchor

Protein folding in the ER

Chaperones
BIP (ER hsp70)
Calreticulin

Calreticulin

hold onto proteins until properly folded and modified (glycoprotein folding)

Glycoprotein folding by...

Calreticulin

Unfolded protein response - UPR

What happens when unusual high level of unfolded protein exist in cells.
Acts as a TSA agent, only allowing properly folded proteins through to the nucleus. If it is improperly folded, it will be sent back through to be refolded properly.

Membrane Lipids

Phospholipid, glycolipids, cholesterol.

Flippase

Phospholipids are synthesized on the cytosolic side of the SER.
Flippases "flip" the phospholipids through the hydrophobic tail regions.

Ceramide

Will be modified into glycolipids & spingomyelin in the Golgi.

ER retrieval signal

Lys-Asp-Glu-Leu (KDEL)

Golgi

Post office of the cell

What happens in the Golgi?

Lipid metabolism
- Synthesis of Sphingomyelin
- Addition of sugar residues to creamed to make glycolipids.

Protein glycosylation
- Processing of N-linked sugars
- Addition of O-linked sugars

COPI

ER --> Golgi
Backwards to return cargo to earlier compartments

COPII

Golgi --> ER
Coated vesicles
Secretory proteins

Clatherin-coated vesicles

Both directions Golgi

AFR1

Small G-protein family
Formation of COPI and clathrin coated vesicles

SAR1

Small G-protein family
Formation of COPII coated vesicles

Man-6-PO4 receptor

Signal that is recognized to incorporate lysosomal proteins into clatherin coated vesicles

Vesicle fusion

Vesicle gets to its target membrane

v-SNARE on Vesicle
t-SNARE on Target

Requires Rab - small G-protein with GTP bound and energy

Exocyst

8 subunits
Serves to direct vesicles after the Golgi complex to specific locations on the plasma membrane and to mediate their tethering and localization to the membrane immediately before fusion.

Lysosomes

Digestive systems of the cell.

Exocytosis

Synthesize inside of the cell; deliver to outside of cell.

Endocytosis

Synthesized by extracellular fluid; delivered to inside of cell.

The sequence Lys-Asp-Glu-Leu (KDEL) serves to retain proteins in the ER by ...

Binding to receptors within the membranes of the ERGIC and Golgi, which retain them or return them to the ER.

Cargo proteins are transported through the Golgi apparatus ...

By cisternal maturation in which the cistern themselves are the carriers for cargo transport through the Golgi apparatus.

The trans Golgi network is ...

The exit part of the Golgi where fusion of vesicles from the ER occurs.

Which of the following is not involved in the specific targeting of proteins to lysosomes?

N-linked glycosylation in the ER.

Which of the following lipids is/are synthesized in the Golgi apparatus?

Glycolipids

Clathrin-coated vesicles are involved in ...

The uptake of extracellular molecules by endocytosis and the transport of molecules from the trans Golgi network to the lysosomes.

The major model of vesicle fusion holds that the specificity of a vesicle fusing with its target membrane lies in the interaction of pairs of proteins called v-(vesicle) and t-(target) ...

SNAREs

Which of the following is not one of the classes of proteins that regulates the activity of Rab GTPases?

GTF (guanine nucleotide trypolysis factor)

IS = GDI, GDI-displacement factor, & GEF

Which of the following would you expect to find at high concentrations in lysosomes?

Degradative enzymes

The process by which cells degrade their own components by enclosing them in a cytosolic membrane is ...

Autophagy

Calreticulin assists in folding glycoproteins that contain one ______ residue.

Glucose