Protein Trafficking

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Protein Trafficking

• Paul D. Brown, PhD
• paul.brown_at_uwimona.edu.jm
• BC21C Molecular Biology I

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Learning Objectives

• Describe the various ways in which proteins can
  be sorted/translocated
• Co-translational translocation
• Post-translational translocation
• Know the different mechanisms by which proteins
  leave the cytosol and enter the
• nucleus
• mitochondria
• ER
• Golgi network
• The role of signal sequences in this process
• The fate of protein whose sorting signal was
  mutated
• The secretory pathway

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Overview of sorting of nuclear-encoded proteins
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Protein translocation systems

• IM, inner membrane IMS, inner membrane space P,
  periplasm
• OM, outer membrane TL, thylakoid lumen TM,
  thylakoid membrane
• SecYEG, Sec61, TOM, TIM, TOC are protein subunits
  of the translocation systems
• Adapted from Schatz and Dobberstein, Science 271,
  1519 (1996)

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Protein pathways

• Proteins released into cytosol
• Processed to contain specific uptake-targeting
  sequences (later removed by proteases)
• Imported into mitochondrion, chloroplast,
  peroxisome, nucleus
• Mitochondria and chloroplasts contain organelle
  DNA, which encodes organelle rRNAs and tRNA, but
  few organelle proteins
• Most mitochondrial and chloroplast proteins are
  encoded by nuclear genes, which are translated by
  cytosolic ribosomes and then imported
• May be sorted to other organellar compartments
  (second signal sequence)

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Specific uptake-targeting sequences
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Uptake-targeting sequences of imported
mitochondrial proteins
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A cell-free system for studying
post-translational uptake of mitochondrial
proteins
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Translocation into mitochondria

• Cytosolic chaperones deliver proteins to
  channel-linked receptors in the mitochondrial
  membrane
• Delivery of preproteins depend on Hsp70/Hsp40 or
  MSF
• Uptake of mitochondrial proteins requires energy
  ATP in the cytosol, the proton-motive force
  across the inner membrane, and ATP in the matrix

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Translocation into chloroplasts

• Toc components mediate translocation (Toc75 is
  the translocon) Hsp70/ Hsp40 may be involved
• Hsp70 in both the IMS and the stroma assist the
  threading of the preprotein into the chloroplast
• An Hsp100 chaperone also called ClpC (AAA ATPase)
  also binds preproteins in the stroma
• Hsp70/chaperonin (Cpn60) may assist
  folding/assembly of newly-imported protein
• Import into thylakoids (used for respiration)
  uses the SRP pathway

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Translocation into peroxisomes

• Targeting of proteins is initiated
  post-translationally by Pex5/7 proteins, which
  bind the peroxisomal targeting signal (PTS), SKL
• Translocon is Pex14p receptor
• Gated pore that is regulated by membrane
  proteins?
• First organelle demonstrated to import proteins
  without a PTS, by virtue of assembly with other
  proteins that contained a PTS
• Various protein oligomers are imported into
  peroxisomes
• Peroxisomal protein import is defective in some
  genetic diseases, e.g., Zellweger syndrome

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Protein pathways

• Secretory pathway
• Ribosome/mRNA/protein complexes are directed to
  rough endoplasmic reticulum by ER signal sequence
• After translation, proteins move via transport
  vesicles to Golgi apparatus
• Packaged (disulfide bonds, addition of
  carbohydrates, proteolytic cleavages, assembly
  into multimeric units) proteins directed to cell
  surface (secretion), lysosome, or plasma membrane

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The rough ER is an extensive interconnected
series of flattened sacs
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Secretory proteins are found in the ER lumen
immediately after synthesis
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Overview of the secretory pathway

• Cisternal progression
• New proteins in ER lumen or membrane incorporated
  into vesicles, which fuse with cis-Golgi or with
  each other
• Migration from cis- to trans-Golgi
  (localization), and undergo modifications
• Some remain, while others move via small vesicles
  to cell surface or lysosomes
• Secretion may be regulated or constitutive

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Many experiments on the secretory pathway have
relied on cells specialized for the secretion of
certain proteins
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Analysis of yeast mutants defined the major steps
in the secretory pathway
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Translocation into the ER
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Topologies of some integral membrane proteins
synthesized on the rough ER
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Most nominal cytosolic transmembrane proteins
have an N-terminal signal sequence and an
internal topogenic sequence
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Multipass transmembrane proteins have multiple
topogenic sequences
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After insertion into the ER membrane, some
proteins are transferred to a GPI anchor
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Post-translational modifications and quality
control in the rough ER

• Newly synthesized polypeptides in the membrane
  and lumen of the ER undergo five principal
  modifications
• Formation of disulfide bonds
• Proper folding
• Addition and processing of carbohydrates
• Specific proteolytic cleavages
• Assembly into multimeric proteins

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Golgi and post-Golgi protein sorting

• Sequences in the membrane-spanning domain cause
  the retention of proteins in the Golgi
• Different vesicles are used for constitutive and
  regulated protein secretion
• Proproteins undergo
• proteolytic processing
• late in maturation

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Receptor-mediated endocytosis generally occurs
via clathrin-coated pits and vesicles
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The LDL receptor binds and internalizes
cholesterol-containing particles
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The endocytic pathway delivers transferrin-bound
iron to cells
Transcytosis moves some ligands across cells
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At least three types of coated vesicles transport
proteins from organelle to organelle
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Translocation in bacteria

• Two major pathways for translocation in bacteria
  Sec and SRP pathways
• Both converge at SecYEG translocon and use SecA,
  a peripherally-bound ATPase that supplies the
  energy for translocation
• SecB binds to nascent chains containing a signal
  sequence and maintains the preprotein in
  translocation-competent form, then binds SecA
  SRP docks with membrane receptor, FtsY (simpler
  homologues of eukaryotic SRP and SRP receptor)

• Archaea lack SecB, have SRP/FtsY but no SecA
  what drives translocation?
• Archaeal SRP, FtsY, SecYEG more closely related
  to eukaryotic proteins (SecYEG)