Lecture 7 Flashcards
How do materials move within the endomembrane system?
materials (e.g., lipids, proteins, etc) move within endomembrane system via biosynthetic, secretory, and endocytic pathways and
transport vesicles
Endoplasmic reticulum is the…
- starting point for both secretory and biosynthetic pathways
*site of protein (& lipid) synthesis, protein folding, and processing/quality control
Endoplasmic reticulum lumen is made up of….
- lumen - aqueous space inside ER tubules and cisternae
- tubules and cisternae shapes mediated by reticulons
*ER integral membrane proteins - possess unique ‘hair-pin’ (v-shaped) 2o structure: regulate
ER membrane curvature…and overall shape of ER
Endoplasmic reticulum subdomains: Rough ER and smooth ER
RER – mostly cisterane with bound ribosomes, involved in protein and membrane phospholipid synthesis
SER – mostly curved tubules lacking ribosomes, involved in Ca2+ storage, and hormone synthesis
What is a nuclear envelope?
outer nuclear membrane continuous with RER; contains Nups and attached ribosomes
Mitochondria and Plasma Membrane-Associated Membranes (MAM & PAM)
*ER regions that make direct contact with mitochondria or pm, respectively; involved in membrane protein and lipid exchange
ER Exit Sites (ERES)
- ER regions where transport vesicles bud off enroute to Golgi
R ER: one of two main sites in the cell for protein synthesis
- ‘free’ ribosomes in cytoplasm
* fate of nascent, properly-folded soluble or membrane protein
in cytoplasm….
remains in cytoplasm (e.g., glycolytic enzyme)
or…
*targets (post-translationally) to proper intracellular compartment (e.g., nucleus, mitochondria, chloroplasts, etc) - ER ‘membrane-bound’ ribosomes
* fate of nascent, properly- folded soluble or membrane protein in RER….
*remains in RER or localizes (moves laterally in ER membrane or lumen) to another ER subdomain (e.g., nuclear envelope)
or…
*localizes to other ER-derived organelles (e.g., peroxisomes – nascent organelles bud off from ER )
or…
*targets (via transport vesicles) from ER onto another (post-ER) compartment in endomembrane system (e.g., Golgi, lysosomes, pm, etc.)
Co-translational translocation of soluble protein into RER lumen
Steps:
Steps 1 and 2: in cytoplasm, translation of mRNA on ‘free’ ribosome begins
* N terminus of nascent, growing polypeptide emerges from ribosome contains signal sequence stretch of
8-15 hydrophobic amino acids - RER targeting signal
* exposed signal sequence recognized by signal recognition particle SRP = ribonucleoprotein particle consists of
6 proteins and 1 small RNA
* SRP binds to ribosome and stops protein translation
Step 3:
* SRP targets complex (i.e., ribosome, ‘stalled’ nascent polypeptide, mRNA) to surface of RER * SRP binds to SRP receptor hetero-dimeric ER integral membrane protein complex
* cytoplasmic-facing domains of SRP receptor serve as ‘docking site’ for incoming SRP
* interaction between SRP and SRP receptor strengthened by both binding GTP
Step 4:
* GTP hydrolysis results in release of SRP and SRP receptor (used for additional rounds of import) simultaneously, nascent polypeptide and ribosome transferred to cytoplasmic side of Sec61 translocon
* multi-protein complex consists of several ER integral membrane protein subunits (e.g., Sec61a, b and g) forming ‘hourglass’-shaped aqueous channel:
* transfer of nascent polypeptide-ribosome to Sec61 translocon results in N-terminus of nascent polypeptide inserted into opening of translocon channel
* translation resumes and elongating polypeptide chain continues to pass through translocon channel towards ER lumen passage of growing polypeptide through translocon driven by translation (i.e., co-translational translocation)
Steps 5 and 6:
* as N-terminal signal sequence enters ER lumen cleaved by signal peptidase and degraded ER integral membrane protein (protease) associated with translocon – catalytic domain of signal peptidase faces ER lumen. peptidase recognizes cleavage sequence motif at C-terminal end of signal sequence
* co-translational-translocation of polypeptide into ER lumen continues…
Steps 7 and 8:
* translation completed and ribosome released from translocon
* remainder of nascent protein enters ER lumen
* Ttranslocon closes – pore ‘plug’ moves back in (i.e., blocks) aqueous channel
Sec 61 translocon
- ‘hourglass’-shaped translocon channel contains ‘pore ring’ ring of 6 hydrophobic amino acids located at narrowest diameter of channel serves as gate’ to seal channel to ions/small molecules (including during protein translocation) – helps maintain ER compartmentalization
- translocon channel also blocked by short a-helix ‘plug’ second gate-keeping mechanism (helps maintain ER compartmentalization) during protein translocation growing polypeptide forces ‘plug’ away from channel
membrane protein topology
= number of membrane-spanning domains and orientation
Transmembrane domain (TMD)
= typically a-helix of ~16-25 hydrophobic amino acids (energetically favorable within hydrophobic interior of phospholipid bilayer)
* several different classes of integral membrane proteins synthesized at ER
Type I membrane protein (N ER lumen-C cytosol)
• nascent polypeptide-ribosome complex targets and associates with translocon
• N-terminus of nascent polypeptide enters ER lumen and signal sequence cleaved by signal peptidase (Step 1)
• polypeptide co-translational translocation continues (Step 2)
• eventually first and only hydrophobic TMD enters translocon (Step 3)
• TMD serves as stop-transfer anchor (STA) sequence ‘stops’ further translocation of polypeptide through translocon
• STA moves laterally out of translocon and becomes ‘anchored’ in adjacent phospholipid lipid bilayer (Steps 4 and 5). translation continues, elongating polypeptide extends into cytosol (Step 5) translation completed, ribosome released
• nascent protein diffuses away laterally in ER membrane bilayer (Step 6)
• final membrane orientation: N ER lumen-C cytosol
Type II membrane protein (N cytosol-C ER lumen)
• opposite orientation to Type I membrane protein * no N-terminal signal sequence, possess internal signal-anchor (SA) sequence
• first and only TMD functions BOTH as signal sequence for binding SRP and mediating nascent polypeptide-ribosome complex targeting to translocon (≈ ER lumenal protein) .. and as membrane anchor
Step 1
• SA ‘flipped’ in translocon so N-terminus of polypeptide faces cytosol
• orientation mediated by several +ve-charged amino acid residues located just upstream (i.e., N terminal) of SA +ve-charged amino acids next to SA determines orientation of most (but not all, e.g., Type-I) membrane proteins synthesized at RER “Positive-outside rule”
Step 2
• translation continues, elongating polypeptide extends into ER lumen via translocon also, SA moves laterally out of translocon and becomes anchored in adjacent membrane bilayer
Step 3
• translation completed, ribosome released, nascent protein diffuses away laterally in ER membrane bilayer
• final membrane orientation: N cytosol-C ER lumen
Type III membrane protein (N ER lumen-C cytosol)
• same orientation as Type I membrane protein, but, like Type II proteins, possess internal signal-anchor (SA) sequence (i.e., no N-terminal signal sequence)
Step 1
• SRP-dependent targeting to and insertion into translocon similar to Type II membrane protein, but +ve-charged amino acids located downstream (C-terminal) of SA - i.e., SA not ‘flipped’ in Type III membrane proteins
Step 2
• translation continues, elongating polypeptide extends into cytoplasm
Step 3
translation completed, ribosome released, protein diffuses away laterally in ER membrane bilayer
• final membrane orientation: N ER lumen-C cytosol
