Lecture 3:

Organelles
Today’s class is located
in….
Chapter 12
Chapter 16
WARNING!!!!!

THIS CLASS WILL BE ONE OF

THE MOST BRUTAL CLASSES
OF THE COURSE.

WE COVER MANY UNIQUE

ASPECTS OF ORGANELLES AND
MANY MECHANISMS THAT
OCCUR WITHIN THEM
Organelle structure and
function
 Nucleus
 Nucleolus
 Endoplasmic Reticulum
 Smooth and rough
 Golgi apparatus
 Ribosomes
 Lysosomes
 Endosomes
 Plasma membrane
 Cytosol
 Mitochondrion
 Microvilli, etc.
 Nucleus

Nucleus contains
 Nucleolus:
 Nuclear matrix
 Nucleoplasm
 Chromatin
Nuclear envelope
 Plasma membrane
 Nuclear pores
 Nuclear Matrix: cytoskeletal structure.
Spongelike networks that compartmentalize
and maintain structure. (importance is still
debated)
 Nucleoplasm: Viscous liquid inside the
nucleus.
 Nucleolus
 Chromatin that serves as the site of ribosome
synthesis and assembly.

Noncoding RNAs in eukaryotic ribosome biogenesis and function, Denis L J Lafontaine1,

Tandem repeat arrays
 rRNA, tRNAs, noncoding RNAs, snRNAs for RNA
splicing, histone genes, etc..
 Repeated genes that encode nearly identical
proteins or functional RNAs.
 Why have so many?
 Example.
 Embryonic development cells have doubling time
of ~24hrs and contain 5-10 million ribosomes.
Cannot produce enough rRNA with only one copy
Eukaryote (80s)
60S subunit (large)
5S : 121 nt, 5.8S : 156 nt, 28S :
5070 nt
40s subunit (small)
18S : 1869 nt
Ribosomal RNAs are
made by…..
 RNA polymerase 1 transcribes a 47s
5’
3’

ETS=external
transcribed
spacers
ITS=internal
transcribed
spacers

 What about rRNA 5s?

Proteins are produced by
RNA Pol II
 80 ribosomal proteins
 > 250 assembly factors (AFs)
 Catalyze RNA cleavage
 RNA modifications
 RNP modeling
 Most AF functions unknown
 snoRNAs (small nucleolar RNA)
 Also produced by Pol II in nucleoplasm
 Small abundant RNAs that help folding and
assembly of ribosomes.
 Base pairing of pre-
rNA with snoRNA
inside the snoRNP
(small nucleolus
ribonucleoprotein)
allow the addition of
methyl through the
snoRNP enzymatic
activity
Ribosomal assembly
Translation via
Ribosomes
In cell biology labs that
study RNA…….
 What do we use as a “control” RNA to compare
expression of mRNAs in different cells?
 Housekeeping genes.
 GAPDH
 18s
 There’s a lot of these genes in a cell.
 Constantly transcribed
 Replicates of these genes.
All of this processing in the
nucleus. Mature mRNA
leaves nucleus.
Extremely long mRNA can
have splicing occur during
transcription.
Chapter 12 in the book!!
Covers….
 Splicing and poly adenylation and how specific
RNA sequences help recruit proteins to these
sites.

 We will NOT discuss poly adenylation in detail,

but you can read it in chapter 12 if you want.
Let’s look at splicing in
the nucleus and how it
works.
Heterogeneous nuclear
ribonucleoproteins
(hnRNPs)
Proteins that interact with immature pre-mRNAs are
required for all of this processing.

Functions
 1.Prevent folding of pre-mRNA into secondary
structures that may inhibit its interactions with
other proteins.
 2.May associate with the splicing apparatus.
 3.Transport of mRNA out of the nucleus.
Which hnRNPs are important
for splicing, folding, etc.?
 Specific protein domains interact with specific
sequences of RNA located throughout pre-
mRNA
 Example: sequencing located on introns will
recruit hnRNPs associated with splicing.
 mRNA splicing
 2 transesterifications take place to remove intron and
ligate two exons together.
Consensus sequence in most
exons and introns help
recruit Spliceosome complex.
30-40 nucleotides at the end are important. Rest of
intron is not important for splicing
How does that sequence
recruit splicing factors?
A group of proteins and RNAs recognize each other and
the pre-mRNA sequences
 Small nuclear RNAs (snRNA)
 Five U-rich small nuclear RNAs (snRNAs),
designated U1, U2, U4, U5, and U6, participate in pre-
mRNA splicing.
 Ranging in length from 107 to 210 nucleotides, these
 Small nuclear ribonucleoprotein particles
 6 to 10 proteins in the nucleus of eukaryotic cells.
snRNAs U1 and U2 recognize
pre-mRNA sequence.
Once U1/U2 bind, the U4,U5,U6 snRNP
complex of RNAs and proteins bind to
the U1 and U2 snRNAs.

 A spliceosome is formed
with all of these
subunits
First Transesterification
occurs.
 U1 and U4 snRNs
(including RNA and
Proteins associated with
them) are released after
spliceosome has been
formed and intron is
ready to be removed.
 Then first
transesterification occurs.
Then second
transesterification occurs
Question: What about
alternative spicing? How do
we know which unwanted
exon needs to be removed?
Splicing silencer or
enhancer sequences on
pre-mRNA
 Different sequences on the pre-mRNA names
intron splicing silencer (ISS) or recruit other
hnRNPs that stop U2/U1 from splicing out the
intron with the adjacent exon on that side.
 It will instead “skip it” and splice with the next
exon.
Other sequences help promote splicing
 Intron splice enhancer
 ISE
 Exon splice enhancer
 ESE

Intron Exon
Understand!!!!!! Different
sequences on pre-mRNA are
recognized by different
snRNPs to help decide
whether certain exons should
be spliced out or not to
generate different isoforms of
the “future” protein.
Are these processes
compartmentalized in
the nuclear matrix?
Maybe.

 A). Red= poly adenylation

 Blue = chromatin
 B). Yellow = splicing factor
Nuclear membrane
 Inner and outer membrane
are fused with nuclear
pores
 The outer nuclear
membrane is continuous
with the rough
endoplasmic reticulum
Nuclear import and
Export
Export
 RNAs
 microRNA
 tRNA
 mRNA
Import
 Proteins
 Transcription Factors for gene control
 mRNA splicing proteins
Import proteins into the
Nucleus
 Importin binds to
the NLS
 Nuclear localization
sequence (NLS)
 Many transcription
factors have this.
 Kinases and
phosphatases can
change
conformation and
open NLS for TF to
enter nucleus
Exporting proteins
Exporting RNA
Specificity:
Different Cargo proteins
 Exportin 5
recognizes, captures,
and transports
miRNAs across the
pore.
 Exportin 1 performs a
very similar process
with tRNA.
mRNA Export

P514 Textbook
Endoplasmic Reticulum
(rough:RER)
 An extension of the Nuclear membrane
 Outer membrane becomes RER
 Ribosomes scattered throughout membrane

The nuclear envelope and inner-nuclear-membrane-protein sorting during interphase Iain W. Mattaj
Nature Reviews Molecular Cell Biology 5, 65-69 (January 2004)
Key roles for
Endoplasmic Reticulum
 Network of membrane-enclosed tubules and sacs
(cisternae)
 Usually the largest organelle in eukaryotic cells
 Membrane may account for half of all cell membranes.
 Rough Endoplasmic Reticulum (RER)
 Has many ribosomes on the surface
 Protein processing
 Smooth Endoplasmic Reticulum (SER)
 Not associated with ribosomes
 SER DOES HAVE CELL SURFACE PROTEINS THAT FUNCTION
AS ENZYMES!!!
 Lipid metabolism
A beautiful Secretory pathway

 Nucleus: DNA-RNA
 RER: RNA-Protein
 Golgi: Processing and Secretion

The Cell: A Molecular Approach. 2nd edition. Chapter 9

Cooper GM.
Sunderland (MA): Sinauer Associates; 2000
Experimental Design
 RER vs Cytoplasmic
Translation
 RER
 Proteins destined for secretion,
the ER, Golgi, lysosomes, or
the plasma membrane.
 Secretory proteins
 Surface receptors, enzymes
and channels.
 “Free” ribosomes
 Proteins to be incorporated into
the nucleus, mitochondria,
chloroplasts, or peroxisomes.
Targeting proteins to
Endoplasmic Reticulum

 Targeted during synthesis or after their

translation
 Humans (mostly during synthesis)
 Yeast (both)
 Signal Sequence
 Basic AA (Arg) followed by
many hydrophobic AAs.
 Signal Recognition particle
 7SL RNA ~30nt
 Many proteins
 Bind to Signal sequence
with hydrophobic binding
groove
 Interaction with SRP
receptor RER membrane
proteins
Membrane bound
insertions (Ch.16)
Cleavage of SRP,
Insertion into membrane
(Type I)
Type II &III
transmembrane insertion
 Non cleavable
 Alpha helix is
exported into the
cytosol
 N terminal is now
outside or inside
depending on
orientation of
signal sequence

 ~ Pg 660 Textbook
+++ charge amino acids
dictate direction of
insertion
 Type 2
What direction do I go?

 STA: Stop transfer anchor, SA: signal-anchor

 +++: positively charged amino acids (go on Cyto side)
 ~ Pg 660 Textbook
Type IV insertion
Internal folding

 Positive numbers = hydrophobic

 Negative number = polar AAs
Chaperones
 Molecular Chaperones
 Proteins that assist in covalent bonding, folding or
unfolding, and assembly or disassembly of
macromolecular structures.
 Prevent newly synthesized chains from aggregating into
nonfunctional structures.
 Sometimes called heat shock proteins
 Heat causes disassembly of proteins
 Chaperones respond and prevent protein aggregates.
 Animal ER chaperones
 GRP78/BiP
 GRP94 (folding of integrins and toll like receptors:
immune)
 GRP170
Post translational transport

 Common in yeast
 Chaperone Hsc70 family member BiP
 BiP: binds to peptide via ATP.
 Prevents sliding backwards
 Prevents folding
 Slowly exchange ADP for ATP
spontaneously. Folding then occurs
Modification in the ER

 Addition of carbohydrates (glycosylation)

 ER and Golgi
 Formation of disulfide bonds S-S
 Proper folding
 Proteolytic cleavages
Glycosylation

 Usually to the Serin/threonine residues

 O linked
 Or to the nitrogen of asparagine
 N-linked
 Most likely promote folding and stability of
glycoproteins
 And sometimes recognition
N-Linked precursor in ER
Disulfide bonds
protein disulfide isomerase
Postranslational
modification in RER
Adding hydrophobic
chains to proteins
 3 classes
 GPI Anchor
 Acylation
 Prenylation
RER also attached lipids
to the protein: GPI
Anchors
 glycosylphosphat
idylinositol (GPI)
anchors
Smooth ER and Lipid
Synthesis
 Lipid synthesis
Membranes have:
 Phospholipids
 Glycolipids
 cholesterols
Flippases
Cholesterol also
synthesized at the ER
and cytosol
Don’t Memorize!!!!!
 Don’t Memorize
Steroids
 Mitochondria and ER
 Multiple Cytochrome
p450 proteins modify
cholesterol
 Oxidation!!
 Just like respiration
and generation of
ATP
 Diff p450 enzymes
generate different
steroids

http://healyourselfathome.com/SUPPORTING_INFORMATION/
CELL_MESSENGERS/HORMONES/STEROIDS/
Golgi Apparatus

 Further processing and glycosylation

 Secretory pathway. Transporting proteins
Mitochondria
Protein import into the
Mitochondria
 95% of mitochondrial proteins are synthesized
on free cytosolic ribosomes
 Some Oxidative phosphorylation proteins
 Mitochondria metabolism proteins
 Proteins for Transcription, translation, replication
 Targeted by mitochondria by amino-terminal
sequences of 20-35 amino acids:
presequences
 Multiple positive charged amino acids in
amphipathic alpha helix structure.
How to transport proteins
into mitochondria?
Need
 Outer membrane receptors and Translocons on
both inner and outer membrane
 Proton motor force via H+
 Mechanism is not currently understood
Entry into the matrix and
folding.
 Tom Complex
 translocation across the
outer membrane
 Tim Complex
 translocase of the inner
membrane
 Heat shock proteins act
as chaperones to
properly fold the protein
Stop transfer sequences
can determine location
 Tom 40

 Tom40 is
general import
pore
 All known
proteins enter
this way

Pg 680-690
 Pg 688-693 of textbook has great example of
this in detail and different Tom and Tim subunits
Mitochondrial DNA

 Evolved from bacteria the eventually developed

a symbiotic relationship with larger cells:
endoxymbiosis
 Usually circular DNA molecules just like bacteria
 Multiple copies per organelle
 Human and animals: ~16kb
 Yeast:~80kb
 Plants: more than 200kb
 Larger ones are mainly noncoding sequences.
Human mtDNA
 13 known proteins encoded by hmtDNA
 16s and 12s rRNA
 22 tRNAs
 These are used for mitochondria translation
 Extreme wobble effect in order to translate with only 22 tRNA
 Normal Translation uses at least 30 tRNAs
Wobble effect compared

Normal tRNA Mitochondria tRNA

tRNA anticodon
U recognizes all
4 nucleotides
 D loop: origin of
replication
 Roman letters
correspond to
respiratory complexes
 Letters represent amino
acids the tRNA will
encode
 DNA mutations in
mitochondria can lead
to ATP synthesis defects
Making ATP with the
Mitochondria
Ch. 8 Textbook
Do not memorize reactions or chemical
compounds. Just See how it fits in with
mitochondria
 Nicotinamide adenine dinucleotide (NAD)

 Flavin adenine dinucleotide (FAD)

Citric Acid Cycle
Oxidative Respiration in inner
mitochondria membrane:
A general look at these
complexes in regards to
Mitochondria
F0F1 proton pump:
ATP Synthase (F-pumps)
 Bacteria, plants, and animals use the F0F1 pump
for the production of ATP
 ATP synthase domain located in F1 complex
 Points to the cytosolic face
 H+ moves from endoplasmic face to cytosolic
face
 Goal of photosynthesis and Aerobic oxydation
 Generate H+ gradient for production of ATP
Asp 61
Twisting of gamma forces a
conformational change in beta
subunits
Moving NADH into the
matrix. P311 of textbook
Lysosomes, endosomes
and Exosomes
 Endosomes are vesicles generated from
endocytosis
 Lysosomes digest cell materials
 Main digestive system
 Peroxisomes process molecules using oxygen

 Today: discuss the interior of these organelles.

 Next week: discuss sorting and movement.
Endosomes fuse with
lysosomes to begin
degradation
 Maintain pH of 5 (100 times more acidic than
cytoplasm
 Lysosomes contain about 50 different
degradative enzymes
 Hydrolyze proteins, polysaccharides, lipids, DNA,
and RNA
 Mutations responsible for 30 different human
genetic diseases
 “Lysosomal storage diseases”
 These enzymes are acid hydrolases
 They are active at pH 5
Why active at pH 5 only?
How do we make it so
Acidic?
 Have to pump protons in the
vacuole
 V-class pumps
 Paired with Cl- Channels to stop the
electronic potential from forming
 Pumps described in detail on pg 252
Phagocytosis and
autophagy
 Phagocytosis: taking up large particles such as
cell debris, bacteria, aged cells
 Autophagy: literally means “eating oneself” will
discuss this with cell death and life lecture.
Peroxisomes
 50 different enzymes
 Oxidative reactions that lead to production of
hydrogen peroxide.
 Detoxify the cell (liver and kidney cells)
 Catalase: decomposes hydrogen peroxide by
converting it to water or organic compounds.
 Breaks down
 Uric acid
 Amino acids
 Fatty acids
Formation of
Peroxisomes
 Form like mitochondria
 Proteins are imported as after
translation
Pex receptor recognizes
peroxisome targeting sequence.
Peroxisomes for energy

 Yeast and plants use

this more than
humans, but humans
also break down fatty
acids for energy
metabolism via
peroxisomes
Plant seeds contain
glyoxisomes
 Very similar to peroxisomes
 Main goal to break down stored lipids as a
source for carbon and energy for growth.
 Has additional enzymes for glucose precursors
from fatty acids.
Pg 181 Textbook
Reading Material

 Nucleolus:
 Nuclear pores: Unit 12.3 Textbook
 RNA Export: Exporting RNA from the nucleus to the
cytoplasm Alwin Köhler & Ed Hurt Nature Reviews Molecular
Cell Biology 8, 761-773 (October 2007)
Endoplasmic Reticulum
 Ch 16 textbook
 The Cell: A Molecular Approach. 2nd edition. Chapter 9Cooper
GM.Sunderland (MA): Sinauer Associates; 2000 Chapter 9
Mitochondria
 The Cell: A Molecular Approach. 2nd edition. Chapter 9Cooper
GM.Sunderland (MA): Sinauer Associates; 2000 Chapter 10
 Ch