Lectin Membrane Proteins

Integral Membrane Proteins

  • Integral membrane proteins are permanently attached to a membrane. They are also called membrane proteins.
  • They are lodged in the lipid bilayer, with some parts passing across and others not.
  • The membrane-spanning region is typically an alpha helix.
  • Hydrophobic alpha helices often form membrane-spanning domains.
  • Hydrophobic side chains allow the protein to remain lodged in the lipid bilayer.
  • Targeting to the ER membrane involves the signal recognition particle (SRP), the SRP receptor, and the translocon.
  • Integral membrane proteins are classified based on their topologies (number of times they cross the membrane) and their orientation (whether the N-terminus is in the cytoplasm or lumen).
  • Topology is directed by topogenic sequences, which determine the number of transmembrane alpha helices.
  • The number of topogenic sequences equals the number of transmembrane alpha helices.
  • The charge of the amino acids surrounding the hydrophobic core determines the orientation of the membrane protein.
  • The end of the protein with the greatest number of positive amino acids will end up in the cytoplasm or cytosol.
  • Can be single pass (one membrane-spanning domain) or multi pass (more than one).

Insertion of Single Pass Integral Membrane Proteins

  • A single pass protein can be directed to the ER by an N-terminal signal sequence that is cleaved.
  • The N-terminal start-transfer sequence also functions as the ER signal sequence.
  • As the polypeptide chain is synthesized, the ER signal sequence exits the ribosome and is recognized by the SRP.
  • The SRP binds to the ER signal sequence and the large ribosomal subunit, causing a halt in translation.
  • The ribosome-SRP complex translocates to the SRP receptor in the ER membrane.
  • The presence of the ER signal sequence causes the translocon (Sec61 complex) to open.
  • The SRP and its receptor dissociate, and translation begins again.
  • The protein passes through the translocon, with the ER signal sequence remaining lodged in the membrane.
  • An internal stop-transfer sequence (anchor sequence), also a hydrophobic alpha helix, is present.
  • When the anchor sequence reaches the ER membrane, it remains lodged there.
  • Translation continues, and the C-terminus ends up in the cytoplasm.
  • The N-terminal signal sequence is cleaved by signal peptidase.
  • The protein is now anchored only by the anchor sequence.
  • The translocon opens sideways to release the signal sequence and the protein into the ER membrane.

Single Pass Proteins with Internal Signal Sequence

  • Single pass integral membrane proteins can also be targeted by an internal signal sequence that also acts as the anchor sequence.
  • The protein has only one internal sequence and it acts as both the ER signal sequence and the anchor sequence
  • Orientation in the ER membrane depends on the charge around this topogenic sequence.
  • The SRP recognizes the internal ER signal sequence, binds to it and the large ribosomal subunit.
  • Translation halts and is targeted to the SRP receptor in the ER membrane.
  • The insertion direction is determined by the charge around the topogenic sequence.
  • If more positive amino acids occur before the signal sequence, the N-terminus will end up in the cytoplasm.
  • If a greater number of positive amino acids occur after the topogenic sequence, then the C-terminus will end up in the cytoplasm.
  • Once the charge in the insertion direction is determined, the SRP and its receptor dissociate and translation resumes.
  • Because the ER signal sequence is internal and also acts as the anchor sequence, it remains lodged in the membrane.
  • The ER signal sequence is not cleaved off because it functions as the anchor sequence.

Insertion of Multi Pass Membrane Proteins

  • Polypeptide chains pass back and forth repeatedly across the lipid bilayer.
  • An internal initial signal sequence acts as the ER signal sequence.
  • The SRP is directed to the ER membrane to bind to the SRP receptor in the membrane.
  • The translocon opens.
  • The ER signal sequence is a hydrophobic series of amino acids, forming a membrane-spanning domain.
  • The SRP and its receptor dissociate, translation begins again, and the polypeptide chain passes through the translocon until it hits another internal anchor sequence.
  • In the simplest case, a multi pass membrane protein has two membrane spanning domains, having two internal anchor sequences.
  • Proteins can have more than two membrane spanning domains.
  • Each subsequent membrane spanning domain is inserted as a hairpin through the translocon.
  • There are multiple anchor sequences and topogenic sequences.

Flowchart for Integral Membrane Protein Insertion

  • Translation begins, involving the SRP receptor (same process as in lecture 14).
  • As the polypeptide chain emerges from the ribosome, the SRP recognizes and binds to the ER signal sequence.
  • The SRP binds to the large ribosomal subunit, causing translation to halt.
  • The SRP-ribosome-protein complex translocates to and binds to the SRP receptor located in the ER membrane.
  • The presence of the ER signal sequence causes the translocon to open.
  • The SRP and the SRP receptor dissociate and are reused.
  • Translation starts again but this time, the signal sequence anchors the protein in the membrane.
  • Peptide synthesis continues.
  • Multiple anchor sequences result in multiple membrane-spanning segments.
  • When translation finishes, the protein spans and is lodged in the membrane, and its orientation is determined by the charge around the internal topogenic sequence.

Summary of Signal Sequences and Targeting

  • Single pass integral membrane proteins are targeted to the ER membrane by an N-terminal ER signal sequence.
  • The ER signal sequence is hydrophobic and remains lodged in the ER membrane.
  • An internal anchor sequence is also hydrophobic and remains lodged in the membrane.
  • The N-terminal ER signal sequence is cleaved by signal peptidase, leaving the internal anchor sequence as the only membrane-spanning domain.
  • The N-terminus ends up in the lumen, and the C-terminus ends up in the cytoplasm.
  • Single pass membrane proteins can also be targeted by a single internal sequence that acts as both the ER signal sequence and the anchor sequence.
  • The orientation is determined by the charge around that single internal sequence. The end with a greater number of positive amino acids will end up in the cytoplasm.
  • The internal sequence is not cleaved.
  • Multi pass proteins have multiple internal anchor sequences and an internal ER signal sequence.
  • Each of these anchor sequences and the internal ER signal sequence forms a membrane spanning domain, this means it passes the membrane multiple times.

Insertion into the Plasma Membrane

  • Proteins are targeted to the ER membrane and inserted involving the SRP, SRP receptor, translocon, and signal sequences.
  • Membrane proteins enter the biosynthetic-secretory pathway.
  • Synthesized at the rough ER, inserted in the ER membrane, and transported to the Golgi in transport vesicles.
  • Targeted to the cis-Golgi, pass through each Golgi compartment (cis, medial, trans), undergoing processing.
  • Vesicular transport model describes proteins passing through compartments in vesicles.
  • Maturation model describes cisternae maturing and moving.
  • When reaching the trans-Golgi, they bud off into vesicles targeted to the plasma membrane.
  • Vesicles fuse with the plasma membrane, incorporating the integral membrane protein.

Broad Timeline Summary

  • DNA is transcribed into messenger RNA, the messenger RNA is processed, with quality control via nonsense mediated messenger RNA decay, checked it is spliced correctly, and transported into the cytoplasm.
  • The messenger RNA is translated into protein on the ribosome.
  • Common pool of ribosomes either remain as free ribosomes (synthesizing intracellular proteins) or become membrane-bound at the rough ER (synthesizing specific organelle proteins or membrane proteins).
  • Ribosomes become membrane-bound when the ER signal sequence is recognized by the signal recognition particle.
  • The signal recognition particle takes the protein-ribosome complex to the SRP receptor, which is found in the ER membrane, and now they become membrane bound ribosomes.
  • The newly formed polypeptide chain can pass through the pore in the membrane, the translocon or SEC61 complex.
  • Membrane proteins are inserted into the membrane; those specific organelle proteins that are synthesised by the membrane bound ribosomes end up in the ER, Golgi, endosome, lysosome or are secreted (Lecture 14). They are transported across the ER membrane.
  • Firstly in the ER lumen the proteins fold correctly, assemble into multimeric subunits, and disulfide bonds are rearranged, glycosylation can begin, then special glycosylation via the GPI anchor.
  • Once the protein is properly folded and assembled, it can then be packaged into vesicles and passed to the golgi.
  • In the golgi it passes from the cis golgi through the medial to the trans Golgi, and there are different processings that occur in each of these compartments as the protein moves through these different golgi compartments.
  • When the proteins reach the trans Golgi, they are sorted and packaged into vesicles which then take protein to their desired location. It might be to the plasma membrane so they can be secreted or to the lysosome.