ER TRANSLOCATION / ER FUNCTION

Overview

• Biosynthetic/Secretory pathway
• Constitutive
• Regulated
• Endocytic pathway
• vesicle formation/fusion
• sorting signals/receptors for incorporation into vesicles

Key experimental Approaches

• autoradiography
• pulse/chase
• cell fractionation
• yeast mutants

Key points

• ER stucture/function
• smooth vs rough
• primary: site for secretory protein syn including folding & glycosylation
• site for lipid syn
• Ca sequestration/regulation, detoxification
• ER translocation
• key steps: targeting, channel gating, force generation
• Protein processing
• folding, glycosylation

Examples

• CFTR (defects in folding)

Cytosolic & Membrane Bound Protein Synthesis

• two classes of ribosome
• free
• membrane-bound
• determined by protein being made: signal peptides drive membrane binding
• the ribosomes cycle on & off the mRNA (polyribosome structure)
• two classes of mRNA
• membrane bound: while ribosome subs cycle, mRNA stays ER bound
• free: while ribosome subs cycle, mRNA stays free

Types of Protein Synthesized on Free Ribosomes include:

• cytosolic (glycolysis, cytoskeleton)
• peripheral membrane proteins (spectrins, ankyrins)
• nuclear proteins
• peroxisome, mito, chlor proteins
• (C-terminally anchored proteins)

ER Translocation

• two-classes of translocated protein
• transmembrane protein: embeded in membrane
• water soluble proteins: pass through membrane
• secreted
• retained
• two-types of translocation
• co-translational
• post-translational

Steps for translocation

• exposure of N-terminal signal sequence
• 6-20 nonpolar residues
• shown by =/- signal mRNA and protease protection in vitro trans.
• approx. 60 a. acids span ribosome=>80 for exposure
• may also be internal for some proteins
• binding of SRP
• binds signal peptide and site on ribosome
• ribosome binding triggers GTP exchange
• 6 protein subunits and a small RNA molecule
• translational arrest:
• protects against premature folding of N-terminus
• protects cell cytoplasm from lysosomal enzymes etc.
• SRP binding to SRP receptor
• requires GTP bound SRP and GTP-bound SRP receptor
• ribosome binding to translocon
• insertion of nascent peptide into translocon
• channel opening
• 4-6 nm diameter = huge!
• closed at either end (ribosome, Bip)
• SRP release: translation ensues
• triggered by GTP hydrolyis by SRP and SRP receptor
• translocation
• cotranslational
• chaperone driven
• signal peptidase cleavage
• oligosaccharyltransferase addition of carbohydrate
• folding of translocated domains and quality control
• protein disulfide isomerase
• Bip
• calnexin
• ribosome release
• channel closure

ER background

• major cellular membrane >50%
• interconnected branching tubules and sacs
• ER lumen: interconnected convoluted space= 10% cell volume
• functions in lipid and protein synthesis

Smooth ER

• ribosome-free
• includes transitional ER: sites of vesicle formation
• specialized functions-> large increase in amount of smoot ER
• lipid metabolism: eg cholesterol syn. for steroid hormone production
• lipid synth. for lipoproteins produced in liver (carry lipids in blood to distant sites)
• detoxification reactions: eg cytochrom p450: to solubilize insoluble for excretion
• calcium sequestration: eg sarcoplasmic reticulum
• release of glucose by ER-localized glucose-6-phosphatase
• expansion can be stimulated by drugs such as phenobarbitol
• retraction (upon drug removal) is by autophagocytosis

MEMBRANE BIOSYNTHESIS

• membranes are asymetric
• asymetry maintained during transport
• lumenal ER=lumenal Golgi=lumenal vesicle=external PM
• integral membrane proteins
• stop transfer sequences
• lateral exit from gated translocon
• positive charge common at cytosolic interface of TM domain
• Fig 8.13 shows type I vs type II generation
• lipid synthesis
• most lipids made in ER, except:
• sphingomyelin & glycolipids finished in Golgi
• some mito & chlor lipids are syn. at mito or chlor mem.
• occurs on cytoplasmic leaflet by ER integral enzyemes
• involves some flippase action to move to other leaflet
• move to destinations in cell by transport vesicles
• composition changes during transport
• PC: ER>G>PM, PS & SM: ER<G<PM, due to 3 activities:
• modified during transport by localized enzyme
• sorting (retention/inclusion) during vesicle formation
• phospholipid transfer proteins move lipid from mem to mem via cyto

GLYCOSYLATION (RER)

• carbohydrate addition to most secreted and integral proteins
• two types N and O
• function
• binding sites for interactions
• sequence
• many sugar sequence permutations possible
• but each protein has a characteristic or constant sequence
• glysosyltransferases
• transfer monosaccharide from donor to acceptor
• donor = nucleotide suger (CMP-sialic acid, GDP mannose, UDP NAG)
• acceptor = growing end of CHO chain
• sequence of addition depends (in part) on spatial localization of enzyme
• initial step for N
• dolichol phosphate lipid carrier
• made of >20 isoprene units
• NAG added twice
• mannose added 5 times
• flips to lumenal side
• mannose added 4 times (transfered from a dolichol donor)
• glucose added 3 times (transfered from a dolichol donor)
• transfer to asparagine in NX(S or T)
• initial modification of core occurs in ER
• glucosidase mediated removal of the terminal glucoses
• yields high mann form ready for export