BIOL 331: Advanced Cell Biology

Unit 3, Part 4: Intracellular Compartments and Protein

Sorting, cont..
Chapter 12, 6th Edn pp. 666 675

Mon Jan 23 2017 1

 BIOL 331: Points to Ponder
So far in this unit:
- looking at biogenesis of sub-cellular organelles from the perspective of
protein import - learned how proteins imported into nucleus, mitochondria

Today:
- examine protein translocation into peroxisomes, begin look at import
into ER
- End of todays class (Q. 12-10) will introduce you to another tool

Looking ahead:
- complete Chapter 12/MBoC on Friday (Jan 27);
- in-class review - Friday (~ latter 1/2 of class)
- Monday (Jan 30, whole class);
- MidTerm #1: Wednesday Feb 1, 10:30 11:20, DC 1350

- if youre not having fun yet, today is last day to drop for full fee refund
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 What Are Peroxisomes?
- found in essentially all mammalian
cells
- small vesicular compartments, (0.1
1 m dia.)
- major users of O2 and H2O2

- urate oxidase, catalase; confer

electron density in EM

- urate oxidase found in all

prokaryotes & eukaryotes, but not in
primates - removes uric acid, potent
antioxidant (!); too much uric acid
can cause gout; why did we lose
it?......

- peroxisomal functions are

indispensable for normal human
metabolism

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 Peroxisomes: Functional Overview

Main (not all) functions:

- -oxidation of Very Long Chain Fatty Acids (VLCFAs)
- all FAs ultimately degraded in Krebs cycle (in mitochondria,
via carnitine esters) to produce ATP
- -oxidation of FAs
- biosynthesis of ether lipids, including plasmalogens (chief
component of myelin!) and platelet activating factor (PAF)
- biosynthesis of cholesterol and other isoprenoidscholesterol
important for formation of bile acids
- detoxification of glycolate to glycine (accumulation of glycolate leads
to precipitation of calcium oxalate in tissues, with deleterious
effects.)

- this is from the perspective of mammals.

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 Peroxisomes in Plants

- two types of peroxisomes in plants

- in leaves, participate in photorespiration
- in germinating seeds = glyoxysomes, convert fat to sugar
- we cant do that..
- glyoxylate cycle AcCoA to succinate, then convert
to glucose in cytosol 5
 Peroxisomal Protein Import
This is where the protein
sorting road map differs
from the previous edition!

Some important points:

1) SKL at C-terminus of
protein functions as signal for
import into peroxisomes

2) some peroxisomal
proteins carry N-terminal
signal sequence

3) proteins do not have to

be unfolded to be imported
into peroxisomes e.g.
thiolase.

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 Peroxisomal Biogenesis
Import not fully understood - involves soluble cytosolic receptors & docking
proteins on cytosolic surface of peroxisome; driven by ATP hydrolysis.

23 proteins identified as participating in peroxisome import - called peroxins;

6 of these form a membrane translocator. One peroxin, Pex5, behaves like
nuclear import receptor, carrying cargo and returning to the cytosol empty.

Peroxisomes may form from ER budding (have distinct protein composition),

but may also arise through growth and fission of pre-existing peroxisomes.

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 A Brief Glimpse @ Peroxisomal Disorders

- 1964, Hans Zellweger described first case of peroxisomal disorder

(aka cerebrohepatorenal syndrome)
- 1973, Sidney Goldfischer reported that kidney and liver tissues
from Zellweger syndrome (ZS) patients had no detectable
peroxisomes

classification: (1) disorders of peroxisome biogenesis (PBD)

- organelle is abnormally formed, missing several functions;
and (2) single-enzyme deficiencies (intact peroxisomes)

combined incidence of peroxisomal disorders is > 1:20,000

ZS is the most severe (incidence ~ 1:50,000); death usually within

first year; incl. mutations in PEX5

X-linked adrenoleukodystrophy (X-ALD) forms largest subset;

childhood cerebral form leads to total disability in first decade

survival in other patients may extend into second and third

decade
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 The Endoplasmic Reticulum (ER)

All eukaryotic cells have

endoplasmic reticulum.

ER lumen = ER cisternal
space

All proteins found on cell

exterior, secreted to
exterior, as well as those
destined for ER lumen,
Golgi, or lysosomes, are
initially targeted to the ER.

Major site of protein

synthesis; also
lipid synthesis & intracellular
Ca+2 storage
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 The Endoplasmic Reticulum (ER)

More than half the total membrane of the average animal cell is associated
with the ER. The ER is a netlike labyrinth of branching tubules and
flattened sacs, continuous with the outer membrane of the NE.
(A)Animal cell, in culture, GFP fusion of ER membrane protein
(B)Plant cell, expressing flu protein in ER 10
 The ER is Structurally Diverse
Regions of the ER are specialized - the extent/nature of these specialized
regions reflects cellular specialization.

Rough ER (rER) is studded with ribosomes, while smooth ER (sER) has no

ribosomes attached; most cells have less sER than rER. The sER has sites
where transport vesicles destined for the Golgi bud off, called transitional ER.

Network of
tubules, 30
60 nm dia

20 30 nm wide

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 The ER is Functionally Diverse
Cells that secrete a lot of protein likely to have extensive rER.

Cells involved in lipid metabolism, such as synthesis of steroid hormones

from cholesterol, have expanded sER. Hepatocytes produce: 1) lipoprotein
particles; 2) enzymes involved in detoxification reactions (cytochrome P450
family) machinery for production housed in sER membrane.

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(pancreatic exocrine cell) (testosterone-secreting Leydig cell)
 The ER in Muscle Cells: Specialization

Regions of the ER are specialized..

Muscle cells have abundant sER,

called sarcoplasmic reticulum. Ca2+
release into cytosol & re-uptake in
response to extracellular signals via
SERCA trigger myofibril contraction &
relaxation.

http://www.mcatzone.com/uploads/gloss/
sarcoplasmic_reticulum.jpg
http://www.rpi.edu/dept/bcbp/molbiochem/ 13
MBWeb/mb1/part2/images/serca.gif
ER can be Isolated as Microsome Fragments
Homogenization of cells breaks up ER into small fragments that reseal
into small (100 - 200 nm dia.) closed vesicles and are easily isolated by
density gradient centrifugation.

Due to differences in density, it is possible to separate microsomes

derived from sER and rER. rER microsomes are unambiguously rER-
derived, but smooth microsomes are more ambiguous maybe from
Golgi, endosomes, mitochondria.

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 ER Import is Co-translational
Most import into the ER occurs via transmembrane transport in a co-
translational manner. As the polypeptide chain is made on the ribosome, it
is passed/threaded through the ER membrane. (vs. post-translational for
nuclear, mitochondrial, chloroplast, peroxisomal)

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 Signal Sequences and rER Import
Transmembrane proteins - Gunter Blobel, Nobel Prize, 1999
embedded into ER membrane.
Remain as part of the ER or
eventually transported to
plasma membrane or
membranes of other organelles.

Water-soluble proteins -
translocated across ER
membrane into the lumen.
These proteins are destined to
be secreted or sorted to lumen
of another organelle some will
remain in ER.

All of these proteins, regardless

of their final destination, are
first directed to the ER
membrane by an ER signal
sequence. http://nobelprize.org/nobel_prizes/medicine/laureates/1999/illpres/
protein.html
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 Signal Hypothesis & Cell-Free Translation
Signal sequences were first
discovered in cell-free
translation experiments. When
microsomes were not present,
a secreted protein was found
to be slightly larger than that
normally secreted from cells.
The difference in size was
attributable to the removal of
an N-terminal leader peptide.

These observations lead to the

signal hypothesis: the leader
peptide is a signal sequence
that directs the secreted
protein to the ER membrane
and is then clipped off by a
signal peptidase before the
polypeptide chain is completely
translated. 17
 The Signal-Recognition Particle (SRP)

As it emerges from the

ribosome, the leading ER
signal sequence is
recognized and bound by
the signal recognition
particle (SRP).

The SRP is a complex,

composed of 6 different
proteins and one small RNA,
which cycles between the
ER membrane and the
cytosol. The SRP binds to an
SRP receptor located on the http://nobelprize.org/nobel_prizes/medicine/laureates/1999/
ER membrane. illpres/protein.html

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 Signal Sequences & the SRP
ER signal sequences vary greatly in primary AA sequence, but all have a
central stretch of 8 or 9 hydrophobic AAs. The SRP protein structure
reveals a large hydrophobic pocket lined with Met residues (unbranched
flexible side chains), creating a flexible pocket able to bind many different
sizes/shapes of hydrophobic signal sequences.

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 The SRP and The Ribosome

One end of SRP binds the signal

sequence as it emerges from the
ribosome, the other end blocks the
elongation factor binding site of the
ribosome - this inhibits further
translation of the message.

In this way, there is a safety system to

ensure that proteins destined for the
ER are not produced in the cytosol
(especially bad if mRNA encodes a
hydrolase destined to be secreted
from cell). This also prevents
(mis)folding of protein and avoids the
need for chaperones to maintain
unfolded state.

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The Signal, The SRP and The SRP Receptor
Once bound, the SRP + signal sequence & ribosome bind to an SRP
receptor, an integral membrane protein complex of the ER.

The interaction with the SRP receptor brings the whole assembly to an
ER membrane protein translocator. The SRP and the SRP receptor
are then released and the translocator transfers the growing
polypeptide chain across the membrane.

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 Ribosomes
There are two distinct
populations of ribosomes -
membrane bound and free.

Membrane bound
ribosomes are attached to
the cytosolic surface of the ER
and are engaged in the
business of translating a
protein that carries an ER
signal sequence.

Free ribosomes are not

attached to any membrane
and synthesize all other
proteins encoded by the
nuclear genome.

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 Ribosome Cycling in the Cytoplasm

The ribosome
subunits are
generic (non-
specific) and can
be used to
translate either
type of protein.
Several ribosomes
can attach to a
single mRNA
molecule at any
given time - the
complex of mRNA
+ several
ribosomes is called
a polyribosome
(or polysome).

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 OVERVIEW

What are the primary roles of peroxisomes?

What is a signal sequence for targeting proteins to peroxisomes? Is this protein

import co-translational or post-translational? Does the protein need to be unfolded
during transport?

What type of endoplasmic reticulum would predominate in a cell that is actively

making and secreting a lot of protein?

Is ER transport usually co-translational or post-translational? Are there any

exceptions?

What experimental system led to the proposal of the signal hypothesis? What is
the signal recognition particle and what are its dual functions?

What is a polyribosome? Is a ribosome that is attached to the ER membrane

different from a free ribosome?

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 Problem Solving in Cell Biology: Ch. 12

1210
Components of the TIM complexes, the multi-subunit protein
translocators in the mitochondrial inner membrane, are much less
abundant than those of the TOM complex. They were initially identified
using a genetic trick. The yeast Ura3 gene, whose product is an
enzyme that is normally located in the cytosol where it is essential for
synthesis of uracil, was modified so that the protein carried an import
signal for the mitochondrial matrix. A population of cells carrying the
modified Ura3 gene in place of the normal gene was then grown in the
absence of uracil. Most cells died, but the rare cells that grew were
shown to be defective for mitochondrial import.

Explain how this selection identifies cells with defects in components

required for import into the mitochondrial matrix. Why dont normal
cells with the modified Ura3 gene grow in the absence of uracil? Why
do cells that are defective for mitochondrial import grow in the absence
of uracil?

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 Problem Solving in Cell Biology

12-10
Normal cells that carry the modified Ura3 gene make Ura3 that gets
imported into mitochondria. It is therefore unavailable to carry out an
essential reaction in the metabolic pathway for uracil synthesis. These
cells might as well not have the enzyme at all, and they will grow only
when uracil is supplied in the medium.

By contrast, in cells that are defective for mitochondrial import, Ura3 is

prevented from entering mitochondria and remains in the cytosol where it
can function normally in the pathway for uracil synthesis. Thus, cells with
defects in mitochondrial import can grow in the absence of added uracil
because they can make their own.

Reference: Maarse AC, Blom J, Grivell LA & Meijer M (1992) MPI1, an

essential gene encoding a mitochondrial membrane protein, is possibly
involved in protein import into yeast mitochondria. EMBO J. 11, 3619
3628.

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