BIOL 2020 Lecture Notes - Lecture 5: Signal Recognition Particle, Golgi Apparatus, Secretion
Cytoplasmic Membrane Systems
September 23, 25, 28 & 30….
Structure
• See eukaryotic cell internal structure – *dynamic compartments
• Endomembrane system:
o Endoplasmic reticulum
▪ Rough endoplasmic reticulum
• Lumen/cisternal space
• Glycogen granules
▪ Smooth endoplasmic reticulum
o Golgi apparatus
o Endosomes
o Lysosomes
•
Function
• SER Functions
o Synthesis of steroid hormone
o Detoxification – oxygenase cytochrome P450
o Sequestering Ca+
o Leydig cells – testis
o Steroid hormone synthesis
• Rough ER
o Protein secretion
o Lumen proteins
o Membrane proteins
*see Methodology – pg. 273-278
Trafficking
Cell Fractionation
• Breaking cells apart and isolating specific parts of the cell
• This occurs because of microsomes
• Microsomes are not found inside the cell but are found when the cell is homogenized, in
vesicles
Secretion Pathway:
• Pulse Chase Experiment – Demonstrates protein secretion
o Expose cells to radioactive amino acid in a secretory protein for about 3 mins
o Localize protein within cell
o After 3 min, proteins present in rough ER – proteins are probably being synthesized
at rough ER
o Cell is washed with growth medium that contains no radioactive material after 20
mins
▪ Protein moves into golgi apparatus with vesicles attached
o After 120 min, one can see the protein is being excreted from the cell via vesicles
• Cell fractionation
• Autoradiography
Secretory Yeast SEC Mutants
• Yeast with a defective gene that synthesizes proteins but does not secrete the proteins
find more resources at oneclass.com
find more resources at oneclass.com
• The mutants are temperature sensitive
• At a normal temperature, normal secretion occurs
• At a high temperatures,
o proteins may accumulate in the cytosol – indicates transport to the ER defective
o Accumulate in rought ER – defective budding of vesicles from rough ER
o Accuulate in ER to Golgi – defective fusion with golgi
o Accumulate in golgi – defective transport from golgi to vesicles
o Accumulate in secretory vesicles – transport from secretory vesicles to cell surface
defective
• *The pathway can be interrupted at 5 different point
Signal Sequence Hypothesis – how proteins selected for secretion?
• a signal sequence of amino acids is present on nascent polypeptide
• sequence is completely hydrophobic to enter membrane
• the signal recognition particle is composed of 6 proteins and a small RNA which binds to
signal sequence and ribosome
• Consequence: translation (protein synthesis) stops
• Complex moves around and SRP receptor of ER membrane recognizes the SRP binding
together
• Ribosome then binds to translocon
• Translocon – integral protein forms a channel through the membrane
• The signal sequence enters the ER and is incorporated within the lumen of ER while the
SRP is released
• Both SRP and translocon contain GTP
• Once SRP is released, translation can occur again and more proteins can be made
• The protein will elongate, once translation stops ribosome releases SRP
• The signal sequence is removed
• An enzyme associated with translocon cuts the signal sequence away
• BIP or other chaperone pulls protein in through the membrane
find more resources at oneclass.com
find more resources at oneclass.com
Cotranslational Translocation Mechanism
• Integral Membrane Protein
• As the protein moves through and is about to be synthesized in a hydrophobic sequence a
stop-transfer sequence reacts which causes the channel to open
• A protein may have a complex arrangement of several stop-transfer sequences
Membrane Synthesis and Asymmetry:
• A membrane protein can move around cell through secretory
pathway
• Some information from protein may not be secreted
• Proteins synthesized in rough ER get incorporated into
membrane due to stop-transfer sequence
• While they are stopped through vesicles, golgi and rough ER, they
are being modified and synthesized
• Membranes and proteins always demonstrate asymmetry
• Leaflet – one half of bilayer
• Cytosolic leaflet – face into cytosol
• Exoplasmic leaflet – faces into lumen of transport vesicles/rough
ER/golgi
• Intergral proteins in rough ER moves through membrane in same
position or orientation
• What is inside vesicles is exposed to the exterior of the cell
Membrane Lipid Synthesis:
• occurs on cytosolic side of lipid bilayer
Cytosol
Lumen
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
See eukaryotic cell internal structure *dynamic compartments: endomembrane system, endoplasmic reticulum, rough endoplasmic reticulum, lumen/cisternal space, glycogen granules. Smooth endoplasmic reticulum: golgi apparatus, endosomes, lysosomes. Ser functions: rough er, sequestering ca, steroid hormone synthesis, synthesis of steroid hormone, detoxification oxygenase cytochrome p450, leydig cells testis. 273-278: protein secretion, lumen proteins, membrane proteins. Cell fractionation: breaking cells apart and isolating specific parts of the cell, this occurs because of microsomes, microsomes are not found inside the cell but are found when the cell is homogenized, in vesicles. Membrane synthesis and asymmetry: a membrane protein can move around cell through secretory pathway. Intergral proteins in rough er moves through membrane in same position or orientation: what is inside vesicles is exposed to the exterior of the cell. Glycosylation: main modification that occurs to proteins as they are synthesized and move through rough. Er: two types, n-linked, attahces to asparagine with free amino acid in r group, occurs in er and golgi, o-linked.
