membrane proteins 3

membrane biogenesis

• cellular membranes can only be made by expanding pre-existing membranes (not de novo)

• proteins must be sorted during or after translation to their correct compartment or membrane

secretory proteins

• are all inserted into or across the endoplasmic reticulum

  • transport to further compartments (Golgi, PM, endosomes, lysosomes)

  • outer and inner nuclear membranes are continuous with ER

• misfolded proteins are degraded at the ER

  • ubiquitin-proteasome system (back to cytosol)

  • digestion by proteases inside lysosomes (end of secretory pathway)

• ER is protein quality control checkpoint for all organelles in the secretory pathway

rough endoplasmic reticulum

many attached ribosomes, secretory protein synthesis

smooth endoplasmic reticulum

no ribosomes, site of lipid synthesis

targeting signals

• sequences within a protein that specify its organelle location

• are often independent from the structure or biochemical function of the protein

• may be removed by proteolysis after targeting is complete or form part of the native structure

• recognized by their pattern, but usually not exact sequence

steps of targeting

1. recognize a signal on a newly translated protein

   • ribosomes begins translating polypeptide with a signal

   • signal recognition particle (SRP) binds signal and ribosome during translation

2. connect protein to the membrane

   • SRP receptor (membrane protein) binds the ribosome-SRP complex

   • SRP-R links ribosome to translocon pore in ER

3. translocate protein into or across the membrane

   • energy of translation on ribosome drives polypeptide through the translocon

signal hypothesis

• proposed by Gunter Blobel

• observed that newly translated secretory proteins are longer than their final form

• hypothesized that extra sequence is a targeting signal peptide whose main function is to direct insertion into ER

ribosome exit tunnel

• in large subunit, where nascent polypeptides exit

• neutral, polar, too small for tertiary folding

• surface around exit site provides binding sites for ER targeting mechanisms

• 30-40 amino acids of nascent polypeptide between peptidyl-transferase site and exit

signal peptides

• direct proteins to the ER for translocation into or across the membrane, co-translationally

• many secretory pathway proteins have additional targeting signals

  • often polypeptide motif

  • sometimes post-translational modification

• organelles not in the secretory pathway have their own targeting signals / signal peptides

signal peptide pattern

• hydrophobic central region with short polar regions on each side

• in many cases, are at the N-terminus

• shorter hydrophobic region (8-16 residues)

• often cleaved off after translocation

signal anchors

• signal peptides that also become TM helices

• not cleaved off

• can be in different place in the protein (not just N-terminus)

• longer hydrophobic region (18-24 residues)

signal recognition particle (SRP)

• ribonucleoprotein (6 protein subunits, 1 RNA)

• signal sequence recognition subunit with GTPase activity

• translation regulatory domain at opposite end

• RNA strand forms flexible linker

ribosome to SRP

1. SRP samples all nascent polypeptides that emerge from ribosomes

2. when a signal peptide is recognized, SRP attaches tightly to both the signal and the ribosome

   • SRP pauses translation at the ribosome, and binds GTP

SRP-R to translocon

3. the ribosome-SRP complex binds to the SRP-R on ER

4. ribosome moves to the translocon and becomes tightly bound

5. SRP and SRP-R dissociate from ribosome

   • translation resumes, and polypeptide translocates into lumen

   • lumenal polypeptide does not contact the cytosol

SRP and SRP-R (GTP)

• step 2: SRP attached to ribosomes is in the GTP-bound state

• step 3: SPR-R is also a GTPase, and is in the GTP-bound state when it recognizes SRP-ribosomes

• step 5: GTP hydrolysis by both SRP and SRP-R dissociates them and recycles them

ER translocon (Sec61 complex)

• 2 parts that form both sides of aqueous pore

• inactive pore is plugged by part of protein

• active pore is open but tightly sealed onto ribosome (preventing leakage)

• inside of pore is neutral, polar

• the 2 parts of pore open laterally to integrate TM helices into membrane

translocation of lumenal protein

1. signal peptide triggers opening of the translocon

2. polypeptides are translocated in an extended, unfolded state

   • movement of polypeptide is driven by energy of translation pushing it out of the ribosome

3. signal peptidases often remove signal peptide during translocation

   • not sequence specific but has a preferred site

types of TM proteins

• type 1: N-terminus in lumen, C-terminus in cytosol

• type 2: N-terminus in cytosol, C-terminus in lumen

integration of TM helix from protein with N-terminal signal sequence

1. signal peptide starts translocation of lumenal part

2. TM helix is recognized by translocon and integrated laterally into membrane during translation

3. cytosolic part is translated in cytosol

signal anchor integration

1. signal anchor opens translocon like a signal peptide

2. translocon recognizes charges next to the signal anchor to determine orientation in membrane

   • positive charges in cytosol, negative charges in lumen

3. signal anchor is recognized as a TM domain and integrated laterally

multi-pass TM proteins

• combinations of signal anchor and TM helices cause alternating orientation of protein

• topology (TM organization) of secretory pathway proteins can often be predicted

  • hydrophobicity → number of TM helices

  • charge distribution → orientation in membrane

  • disulfide bonds and glycosylation → occur in ER lumen

  • phosphorylation and ubiquitination → occur in cytosol

glycosylation of secretory proteins

• most have oligosaccharides covalently attached

  • help stabilize native state

  • protect against proteases

  • function in cell surface signaling

N-linked (asparagine) glycosylation

• on Asn side chain amide in context of Asn-X-Ser/Thr motif

  • Gln is not recognized by OST

• same glycan always attached at ER

• mostly mannitol with 3 glucose

• attached by oligosaccharyl transferase (OST)

• most N-X-S/T motifs in the lumen are modified depending on accessibility to OST

• glycans can be modified after addition, but not removed until protein is degraded

glycosylation process

1. oligosaccharides are synthesized attached to a specialized lipid in the ER

2. OST attaches glycan during translocation

3. state of glycan is used as a signal in ER quality control of folding

4. glycan is modified in Golgi after exit from ER

ER chaperone system

• ER chaperones

  • BiP (HSP70 equivalent)

    • ERdj proteins (DNAJ co-chaperones)

    • NEF co-chaperones

  • GRP94 (HSP90 equivalent)

    • no co-chaperones

  • thioredoxin family

    • PDI

    • ERp57

• ER N-linked glycosylation

  • calnexin and calreticulin

  • UGGT (UDP-glucose:glycoprotein glycotransferase)

  • glucosidases, mannosidases, lectins (glycan binding)

• ER misfolded protein degradation

  • degradation takes place on cytosolic proteosomes

  • folding is necessary to exit ER to the secretory pathway