Cell and Molecular Biology

Lecture 8

Cytoplasmic
Membrane Systems

John Donnie A. Ramos, Ph.D.

Department of Biological Sciences
College of Science
University of Santo Tomas

The Endomembrane System

‰ Endoplasmic reticulum (ER)
‰ Smooth endoplasmic reticulum (SER)
‰ Rough endoplasmic reticulum (RER)
‰ Golgi complex
‰ Endosomes
‰ Lysosomes
‰ Vacuoles
‰ Transport vesicles
‰ Biosynthetic pathway/secretory pathway
‰ Constitutive secretion
‰ formation of ECM
‰ formation of plasma membrane
‰ Regulated secretion
‰ Endocytic pathway

1
 The Endomembrane System
Characteristics of vesicle movements
‰ Directed transport (pulled by motor
proteins)
‰ Contains sorting signals (molecular
addresses encoded by amino acid
sequences)
‰ Vesicles move by budding and fusion
different organelles

Experiments Used in Membrane Studies

‰ Microscopy
‰ Transmission electron microscopy
‰ Scanning electron microscopy
‰ Confocal microscopy
‰ Fluorescence microscopy
‰ Autoradiorgaphy
‰ Green Fluorescent Protein (GFP)
‰ Biochemical analysis of subcellular fractions
‰ Differential centrifugation
‰ Spectrometry
‰ Electrophoresis
‰ Use of genetic mutant cells (organisms)
‰ Cell-Free Systems
‰ Recombinant DNA/proteins
‰ Sequencing (nucleotide and amino acid)
‰ Cloning and expression

2
 Autoradiography
‰ Used by James Jamieson and
George Palade
‰ Uses radioactive material
‰ Pulse-chase experiment

Green Fluorescent Protein

‰ GFP emits green light (isolated from jellyfish cells - causes
bioluminescence)
‰ Protein of interest can be fused with GFP (chimeric protein)
‰ Can be used to determine the movement of proteins using GFP as tag
‰ Example:
‰ Vesicular stomatitis virus gene (VSVG) fused with GFP (sensitive to
temperature)

3
 Biochemical Analysis

‰ Employs homogenization cells and

isolation of different organelles (cell
fractionation)
‰ Pioneered by Albert Claude and
Christian De Duve
‰ Differential centrifugation

Cell-Free Systems
‰ Isolated ribosomes can synthesize proteins when the needed raw
materials are present in a medium (mRNA, tRNA, amino acids)
‰ Ribosomes secrete out the synthesized protein into the medium
‰ When ribosomes are attached to RER, the synthesized proteins are
not secreted out into medium but into the RER.

4
 Genetic Mutants
‰ Cells or organisms with altered genes (producing mutant proteins)
‰ Results to abnormal function
‰ Example:
‰ Yeast cells with mutant protein (encoded by SEC gene) used for
vesicle fusion (from ER to Golgi complex)

Accumulated
Expanded ER vesicles

Endoplasmic Reticulum
‰ Network of interconnected
tubules
‰ Types:
‰ Smooth Endoplasmic
Reticulum
‰ Rough Endoplasmic
Reticulum
‰ Different sedimentation rates
(basis of separation)
‰ Inner part is called luminal or
cisternal space

5
 Smooth Endoplasmic Reticulum
‰ Functions (in specific cells)
‰ Steroid hormone synthesis
(endocrine cells of gonads and
adrenal cortex)
‰ Detoxification of organic
compounds (hepatocytes)
‰ Secretion of oxygenases (converts
hydrophobic to hydrophilic
substances)
‰ Example: Ethanol, drugs, benzo-
pyrene (carcinogen)
‰ Release of glucose from glucose-6-
phosphate (glycogen in the liver)
‰ Secretion of glucose-6 phosphatase
(phosphorylates G6P)
‰ Collects calcium ions (reservoir of
Ca+)

Rough Endoplasmic Reticulum

‰ Associated with ribosomes (site of protein synthesis)
‰ Ribosomes attached to RER secrete:
‰ proteins needed outside the cell
‰ integral membrane proteins
‰ soluble proteins for endomembrane system
‰ Free ribosomes secrete:
‰ proteins for the cytoplasm (cytoskeletal
proteins)
‰ peripheral proteins of plasma membrane
‰ proteins needed in the nucleus,
mitochondria, chloroplasts,
peroxisomes

6
 Signal Hypothesis
‰ Proposed by Gunter Blobel (1999 Nobel prize winner for medicine)
‰ Secretory proteins contain signal sequence on their N-terminus responsible for entry into RER
cisternal space
‰ Secretory proteins moves into RER cisternal space through translocon (cotranslational
movement)
‰ Secretory signal is 6-15 non-polar amino acids which binds to signal recognition particle ,
SRP, (protein + 7S RNA)
‰ SRP binds to SRP receptor while ribosome binds to translocon
‰ SRP releases signal peptide causing it to enter the RER cisternal space through the translocon

Protein Processing in the RER

‰ Cleavage of signal peptide – performed by signal peptidases
‰ Addition of carbohydrate components (glycosylation) –
performed by oligosaccharyl transferases
‰ Protein folding – performed by molecular chaperones (ex. BiP
and calnexin)
‰ Formation and reshuffling of disulfide bonds – performed by
protein disulfide isomerase (PDI)
‰ Misfolded proteins undergo “reverse translocation” –
move out of the RER through translocon into the
cytoplasm where it is degraded by proteasomes
‰ Assembly of multi-subunit protein complexes

7
 Synthesis of Integral Membrane Proteins

‰ Stop-transfer sequence (STS) - >15 hydrophobic uncharged amino acids

‰ Gating proteins – maintains the permeability of the membrane (causes
the release of ribosome)
‰ STS translocates into the RER membrane

Membrane Biosynthesis
‰ Most membrane lipids are synthesized in
the ER (could be modified along the
biosynthetic pathway)
‰ For integral membrane proteins, N
terminal is located in the luminal space of
ER and C terminal on the cytoplasmic side
(maintained while inside the cell)
‰ During fusion into plasma membrane, the
N-terminal is positioned outside the cell
(extracellular side) while the C-terminal is
on the cytoplasmic side.

8
 Glycosylation of Proteins in the ER
‰ Glycosylation site – specific amino acids sequence where different sugar
molecules form glycosidic bonds
‰ Catalyzed by glycosyltransferases (membrane bound enzymes)
‰ Donor molecules during glycosylation: CMP-sialic acid, GDP-mannose,
UDP-N-acetylglucosamine.

N -linked Oligosaccharide Synthesis

oligosaccharyltransferase

9
Diversification of Oligosaccharides

‰ Evolutionary mechanisms
‰ Quality control
mechanism
‰ Glucosidase – removes
glucose molecules
‰ Glucosyltransferase (GT)
– adds glucose to
misfolded proteins

From ER to GC
‰ Endoplasmic reticulum Golgi
Intermediate Complex (ERGIC)
‰ Vesicular tubular clusters (VTCs)

10
 The Golgi Complex
‰ Identified by Camillo Golgi (1898)
‰ Disklike, flattened, membranous
cisternae with dilated rims and
associated vesicles and tubules
‰ Divided into functionally distinct
compartments
‰ Modification (processing) of
proteins
‰ Trimming of proteins
‰ Modification of amino acids
‰ Modification of glycosylated
proteins

Glycosylation in the GC
‰ Occurs in the cis and medial cisternae
‰ Catalized by different glycosyltransferases
‰ Mostly O-linked glycosylation
‰ Causes variations of proteins

11
 Models of Transport in the GC

Transport Vesicles
‰ Enclosed by protein coats (soluble proteins that
assemble on the cytosolic surface of donor
membranes)
‰ Also called coated vesicles
‰ Functions:
‰ act as mechanical devise that causes the
membrane to curve and form a budding vesicle
‰ Provide a mechanism for selecting the membrane
component to be carried by the vesicle
‰ Types:
‰ COPII-coated vesicles – move materials from ER
to GC
‰ COPI-coated vesicles – move materials from GC
to ER or from cis to trans of GC
‰ Clathrin-coated vesicles – move materials from
trans golgi network to endosomes, lysosomes,
and plant vacuoles

12
 COPII-Coated Vesicles
‰ With 5 protein subunits
‰ Can be blocked by specific antibodies
‰ Can select and concentrate their cargo (materials to
be transported)
‰ Coat proteins bind to signal sequences on the
cytosolic side of ER membrane
‰ The luminal end on transmembrane protein binds
to cargo protein
‰ Example: Sar (a GTP binding protein)

COPI-Coated Vesicles
‰ For the transport of ER resident proteins
that accidentally transported to the GC
‰ Retrograde direction
‰ Contains retrieval signal
(KDEL-lys-asp-glu-leu)
‰ Example: ARF (GTP-binding protein)
‰ Could also be involved in cis-medial-trans
transport in GC

13
 Clathrin-Coated Vesicles
‰ Composed of outer clathrin protein
and inner adaptor proteins
‰ Adaptor proteins connect clathrin
and membrane receptors

Vesicle Fusion
‰ Tethering stage
‰ Rab proteins - GTP binding proteins that
recruits tethering proteins
‰ Tethering proteins – cytosolic proteins
that mediate initial contact between two
membranes
‰ Docking stage
‰ v-SNARE – integral membrane proteins
incorporated into the membranes of
transport vesicles
‰ t-SNARE – integral membrane proteins on
the surface of target organelles
SNARE (Soluble NSF Attachment protein
REceptor)
NSF (N-ethylmaleimide Sensitive Factor)

14
 v and t SNARE Interaction
‰ Formation of four-stranded bundles
comprising of α helices:
‰ 1 syntaxin
‰ 1 synaptobrevin
‰ 2 SNAP-25
The four are attached to
transmembrane helices
‰ Formation of transition water-filled
cavity (center of transmembrane helix
bundle
‰ Fusion of bilipid layers for fusion pore

Exocytosis

‰ Process of membrane fusion and content discharge

‰ Steps:
1. Proteins of 2 membranes make contact with one another
2. Membrane proteins form a closed pore that becomes dilated
3. Diffusion of lipid bilayers along the hydrophobic membrane proteins

15
 Lysosomes
‰ Digestive organelles
‰ Contains ~50 different hydrolytic enzymes (acid hydrolases)
‰ Membranes with highly acidic, glycosylated integral
proteins (prevents digestion by enclosed enzymes)

Lysosomes in
Kupffer Cells

Lysosomes
‰ Variable in size (>1 µm to <25 nm)
‰ Functions:
‰ Digestion of endocytosed material (phagocytes
‰ Acrosome of sperm cell contains digestive enzymes to degrade the
outer covering of ovum
‰ Used in organelle turnover (autophagy) – destruction and
replacement of old organelles

16
 Autophagic Pathway
‰ Autophagolysosome – ER
membrane enclosing an organelle
(e.g. mitochondria) and fuses with
lysosome
‰ Mitochondrion autophagy occurs
every 10 minutes
‰ Increased autophagy when cell is
deprived of nutrients (cannibalism)
‰ Residual body – an
autophagolysosome that completed
its function

Diseases Linked With Defects in

Lysosomal Function
‰ Silicosis (miner’s disease) – uptake of silica fibers (causes leaky
lysosome)
‰ Asbestosis – uptake of asbestos (causes leaky lysosome)
‰ Rheumatoid arthritis – release of lysosomal enzyme from
immune cells into synovial fluid
‰ Lysosomal storage disorders
‰ I-cell disease – absence of hydrolytic enzymes (deficiency
in N-acetylglucosamine phosphotransferase for mannose
phosphorylation
‰ Pompe disease – deficiency of α-glucosidase
(accumulation of undigested glycogen)
‰ Tay-Sachs Disease – deficiency of hexosaminidase A

17
 Plant Cell Vacuoles
‰ Enclosed by a single membrane (tonoplast)
with active transport systems (pumps ions
causing high ionic concentration thus osmosis
occurs)
‰ Temporary storage of ions, sugars, proteins,
polysaccharides, toxins (used for defense)
‰ Site of intracellular digestion (with acid
hydrolases)

Phagocytosis
‰ Cell eating (particles with >0.5 µm in diameter
‰ Phagosome – phagocytosed material enclosed by a
bilipid layered membrane
‰ Phagolysosome – phagosome fused with lysosome
‰ Mode of eating (amoeba and ciliates) or mode of
defense (multicellular organisms)
‰ Opsonins – blood-borne factors (antibodies) that
help enhance phogocytosis by enclosing a particle
‰ Some bacteria uses phagosome to invade the cell
(Mycobacterium tuberculosis)

18
 Endocytosis
‰ Uptake of fluids, dissolved solutes, and
suspended macromolecules
‰ Recycling of membranes
‰ Endosomes – vesicles carrying
endocytosed materials
‰ Types:
‰ Bulk-phase endocytosis
‰ Unspecific uptake
‰ Uptake of any substance near
the plasma membrane
‰ Receptor-mediated endocytosis
‰ Uptake of specific ligands

Receptor-Mediated Endocytosis

‰ Formation of indented portion of plasma

membrane called coated pits
‰ Coated pits concentrates substances to be
endocytosed
‰ Coated pits are covered by clathrin triskelion
(with 3 heavy and 3 light chains)

19
 Cholesterol Uptake
‰ Example of receptor-mediated endocytosis
‰ Low density lipoprotein (LDL) – contains
cholesterol which is an essential component
of the plasma membrane
‰ LDL concentration in blood correlates with
atherosclerosis (narrowing of major arteries)
‰ Atherosclerotic plaques is a site for blood clot
formation (cause of heart attack)

Protein Uptake By Other Organelles

‰ Peroxisomes
‰ Peroxisomal targeting signals (PTS)
‰ Peroxisomal matrix protein - bind to receptors on peroxisome
receptors
‰ Peroxisomal membrane protein signals (mPTS)
‰ Mitochondria
‰ Protein may be delivered to
‰ Outer mitochondrial membrane (OMM)
‰ Inner mitochondrial membrane (IMM)
‰ Intermembrane space
‰ matrix
‰ Chloroplasts
‰ Proteins may be delivered to:
‰ Inner membrane, outer membrane, intermembrane space, stroma,
thylakoid membrane, lumen

20
Proteins Into Mitochondrion

Proteins Into Chloroplasts

21