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 <--> ER

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

In the "pulse-chase" experiment, Palade & colleagues studied the pathway taken by newly secreted proteins in pancreatic acing cells by labeling them with radioactive amino acids & then determining their progressive location within the cells. Which of the

Rough ER --> Golgi Apparatus --> Secretory vesicles

Which of the following is not a possible destination for vesicles leaving the Golgi apparatus?

Mitochondria

In cell fractionation experiments in which sub cellular organelles are separated on the basis of their density, the smooth endoplasmic reticulum is _____ the rough endoplasmic reticulum.

Light than.

The ribosomes of the rough endoplasmic reticulum are targeted to the cytoplasmic side of the membrane via ...

A sequence within the protein being synthesized.

As they emerge from the ribosome, signal sequences are recognized and bound by a(n) ...

Signal recognition particle (SRP).

Which of the following statements about transmembrane proteins is true?

They usually have one or more alpha helices spanning the membrane bilayer.

The lumen of the ER is equivalent topologically to the ...

Extracellular space.

Proteins that span the membrane multiple times have multiple ___ that alternate with multiple transmembrane stop-transfer sequences.

Internal signal sequences.

Protein folding in the ER is assisted by a chaperone called

BiP

Disulfide bonds within or between proteins form easily in which of the following?

ER but not cytosol

The unfolding response involves

a. general inhibition of protein synthesis.
b. increased synthesis of chaperones.
c. increased activity of proteosomes.
*d. All of the above

Most cellular lipids are synthesized in

The endoplasmic reticulum

Newly synthesized membrane lipids are found in both halves of membrane bilayers because they are

synthesized on one surface and flipped to the other surface by proteins called flippases.

The sequence Lys-Asp-Glu-Leu (KDEL) serves as an ER retention signal for proteins by binding to KDEL receptors that

transport the proteins from the Golgi apparatus back to the ER.

Proteins attached to the outer layer of the plasma membrane bilayer are usually attached by a

glycosylphosphatidylinositol (GPI) anchor

Vesicles initially enter the Golgi apparatus by fusing with

the cis (convex) face. They exit from the trans (concave) face.

Which of the following proteins do not pass through the Golgi apparatus?

Ribosomal proteins

N-linked oligosaccharides are added in the

ER and modified in the Golgi.

The plasma membrane of intestinal epithelial cells requires separate targeting of proteins to

two plasma membrane domains: the apical and basolateral domains.

In plant cells, vesicles transport proteins from the Golgi apparatus to

Vacuoles

Vesicles that carry proteins from the rough ER to the Golgi apparatus bud off as
_______ vesicles.

COPII coated

Lysosomal proteins are initially incorporated into _______ vesicles.

Clathrin coated

ARF function on vesicles is regulated by

Binding of GTP

Clathrin coats are bound to specific receptors by a protein called

Adaptor protein

Rothman and colleagues proposed 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

The protein complexes where exocytosis occurs are called

Exocysts

The pH inside lysosomes is about

5.0

Lysosomes digest

a. proteins.
b. nucleic acid.
c. carbohydrates.
d. lipids.
*e. All of the above

Transport vesicles carrying acid hydrolases fuse with

lysosomes.

The process by which cells degrade their own components by enclosure in a membrane derived from the ER is termed

autophagy

The activity of Rab GTPases are regulated by all of the following classes of proteins
except

GTF (Guanine nucleotide trypolysis factor)

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

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

The major site at which membrane lipids are synthesized is

the cytosolic side of the ER membrane.

Calnexin assists in folding glycoproteins that contain one _______ residue.

Glucose

N-linked glycosylation at an Asn-X-Ser/Thr consensus sequence adds approximately
_______ sugar(s) in a single step to the protein.

15

Proteins that span the membrane multiple times have multiple _______ that alternate
with multiple transmembrane stop-transfer sequences.

internal signal sequences

The lumen of the ER is equivalent topologically to

The extracellular space

Which of the following is true of transmembrane proteins?

They usually have one or more ? helices spanning the membrane bilayer.

In yeast, posttranslational translocation of proteins targeting the ER is

a process affecting many newly synthesized proteins.

The ribosomes of the rough endoplasmic reticulum (rough ER) are targeted to the cytoplasmic side of the membrane via

a sequence within the protein being synthesized.

All of the following are contiguous membrane domains except

secretory granules.

Radioactive amino acid-labeled proteins in pancreatic acinar cells were located progressively in

rough ER ? Golgi apparatus ? secretory vesicles.

Mitochondria differ from other organelles such as lysosomes and the Golgi apparatus in that they ...

Contain their own genomes.

In terms of its role in the generation of metabolic energy, the inner membrane in mitochondria is equivalent to which of the following in chloroplasts?

The thylakoid membrane

Each photo center in a chloroplast consists of hundreds of antenna pigment molecules that absorb light and transfer energy to a reaction center.

Chlorophyll

Light is captured by _____ different photosystems associated with the thylakoid membranes of chloroplasts.

Two.

Peroxisomes are involved in all of the following reactions except

The biosynthesis of the enzyme catalase, which breaks down hydrogen peroxide.

The ATP synthases of mitochondria and chloroplasts are examples of ____ proteins in which polypeptide rotation provides a mechanical coupling to ATP synthesis.

Motor

The electrochemical gradient across the mitochondrial inner membrane drives the import of all of the following except ...

Cytochrome c

The human mitochondrial genome encodes only 22 tRNAs. This limited array of tRNAs can read the 64 possible triplet codons through extreme wobble in base pairing at the third codon position & the use of a(n) ____ genetic code.

Nonuniversal.

Human diseases caused by mutations in mitochondrial genomes

are inherited from the mother.

Which of the following is not involved in protein transport into mitochondria?

Vesicular transport.

Cellular metabolism STAGE 1

Digestive enzymes breakdown large molecules to simple subunits.
The breakdown process must act on food taken in from outside, but not on the macromolecules inside our own cells.
Proteins --> amino acids
Polysaccharides -->simple sugars
Fats --> fatty acid

Cellular metabolism STAGE 2

Part 1: glycolysis - breakdown of individual glucose molecules into 2 molecules of pyruvate.
Part 2: pyruvate molecules are imported into mitochondria & converted to acetyl CoA (in the matrix)

Glycolysis

breakdown of individual glucose molecules into 2 molecules of pyruvate.
occurs in cytosol.

Cellular Metabolism STAGE 3

Part 1: Citric Acid Cycle completely oxidizes acetyl CoA to CO2 (in mitochondrial matrix).
Part 2: Electron transport drives synthesis of ATP

Citric Acid Cycle

completely oxidizes acetyl CoA to CO2 (in mitochondrial matrix).
3 molecules NADH
1 molecule FADH2
1 molecule GTP
2 molecules CO2

Inner mitochondrial membrane

Cristae
Where oxidative phosphorylation occurs
Impermeable to ions
70% of mass

Outer mitochondrial membrane

Porins

Inter membrane space (mitochondria)

enzymes that use ATP

Functions of mitochondria

Breakdown of pyruvate to CO2 and ATP
Oxidative reactions
Citric acid cycle
Oxidative phosphorylation
ATP synthesis

Mitochondrial DNA

all rRNA and most tRNA needed to translate proteins are encoded
has its own genetic code (30tRNA would be needed for universal code)
has EXTREME wobble.

Mitochondrial DNA mutations

must be maternal.
if mom has Mt mutation, all offspring will carry it.
degeneration of retinal ganglion cells (RCGs) and their axons is caused by leber's hereditary neuropathy - can be sure that all brothers & sisters will also have disease.

Import of mitochondria proteins with N-terminal presequences

TOM & TIM

TOM

translocase of outer membrane

TIM

translocase of inner membrane

SAM complex

Sorting and assembly machinery

Cardiolipin

a lipid in the inner membrane of mitochondria

Electron Transport

NADH and FADH2 transfer their electrons through a chain of acceptor and donor molecules. Each time the electrons are passed, they fall to a lower energy state and the energy released is used to make ATP.

Anatomy of ATP synthase

flow of protons actually causes it to spin like turbine
mechanically couples P to ADP to make ATP

Chloroplasts

Plastids - proplastid
Have THIRD membrane (thylakoid membrane)

Plastids

2 envelope membrane
- chromoplasts have different pigments (carotenoids = yellow/orange)
Leucoplasts storage, no pigments
- amyloplasts & elaioplasts

What do chloroplasts do?

Fix CO2

Peroxisomes are involved in all of the following reactions except ...

The biosynthesis of the amino acid lysine.

The role of cytochrome c in the electron transport chain is to ...

Transfer electrons from complex III to complex IV

Which of the following is not a protein translocon/translocase found in the mitochondrial inner or outer membrane?

Toc75

Where do phospholipids in mitochondrial membranes such as phosphatidylcholine and phosphatidylethanolamine originate?

In the ER.

What is the major site of energy production in the form of ATP in human cells?

The inner mitochondrial matrix.