Section 3: Protein transport and Disease (Gaucher’s Disease and Cancer as models)
Part A: Co- and post-translational transport
- protein transport is the system whereby proteins reach their correct destinations either within the cell or outside the cell
- as each structure of a cell has a specialized function, each structure must have their own unique set of proteins to allow
them to carry out different functions
- all proteins begin their synthesis in the cytosol so there must be a transport system whereby these proteins reach their
correct subcellular or extracellular comportments
- while many proteins complete translation in the cytoplasm and remain there, others need to reach different locations, such
as the mitochondria or the nucleus; for the nucleus, may be RNA and DNA polymerases
- some proteins more directly to the endoplasmic reticulum while translation is still taking place and then could be
transported to th Golgi apparatus or could remain in the ER
- from the Golgi, could be transported to further locations such as the lysosome or to the extracellular space
Two models of transport: Post- and co-translational
- in post-translational transport, transport of the protein only occurs after translation is completed in the cytosol
- in contrast, in co-translational transport, transport of the newly formed chain occurs while translation is still ongoing
where the entire mRNA ribosomal complex with the nascent protein chain is transported from the cytoplasm to the ER
- co-translational transport is collectively also called the secretory pathway because if a protein is not retained to any of
these subcellular locations, it is ultimately secreted from the cell by default
- note that transport from the ER to the Golgi, as well as subsequent locations is called vesicular transport
How proteins know where to go
- the information to target a protein to a particular destination is encoded within the amino acid sequence of the protein itself
(its primary structure) and these are known generically as targeting sequences or also called signal sequences
- these sequences function as address levels that target a protein for delivery to a particular location
Targeting sequences
- targeting sequences are usually of abut 20 to 30 amino acids in length and could
be contained at the N-terminal region of a protein or at the C-terminal end of the
protein; alternatively, the sequences could be located within the protein sequence
such that it calls on the exterior side of the protein when the protein is folded
- finally, folding of the protein into its proper conformation also gives rise to call
signal patches and signal patches are also used as address labels to target a protein
to a specific location
- for each location and for each signal sequence, there is a set of receptor proteins
that bind either directly or indirectly only to these specific targeting sequences of
the signal patch, this ensures that the information which is contained in the
targeting sequence for a specific location governs the specificity of targeting of the protein
Different targeting signals for different locations
- transport to the ER requires a signal sequence located at the N-terminal and this signal sequence is about 16 to 30 amino
acids in length but importantly, it contains a hydrophobic core of 6-12 hydrophobic aa’s
- some proteins need to be retained in the ER or remain in the ER and these proteins will have a specific sequence at their
C-terminal, known as the KDEL sequence, representing the 4 amino acids
- import into the mitochondria also requires an N-terminal signal sequence
and these signal sequences typically have positively charged amino acids,
but lack negatively charged amino acids whereas ER and the mitochondria
use N-terminal signal sequences, the retention in the ER uses a C-terminal
sequence, import into the nucleus requires an internal sequence of lysine
and arginine residues and import into peroxisomes requires a sequence of
serine, lysine and leucine located towards the C-terminal end
,Protein targeting in the nucleus: the NPC
- protein targeting into and out of the nucleus is an example of post-translational transport, this occurs via the NPC (nuclear
pore complex)
- in eukaryotic cells, the nucleus contains numerous nuclear pores perforating the nuclear envelope and each nuclear pore is
actually an elaborate structure composed of more than 50 different proteins called nuclear porins which give rise to the
nuclear pore complex
- nuclear protein as well as mRNA, tRNA and ribosomes are all transported into and out of the nuclear via the NPC
- we will focus specifically on the mechanisms of nuclear import and export of proteins
Nuclear Protein Import
- step 1: these proteins need to be imported and are called cargo proteins and they
carry a nuclear localization signal (NLS)
- the internal lysine and arginine sequences (NLS) is recognized by importin which is
a nuclear import receptor that is located inside the cytosol
- step 2: once importin has bound the NLS on the cargo protein, the importin-cargo
complex then diffuses through the NPC into the nucleus of the cell
- step 3A: once inside the nucleus, a GTP-binding protein called Ran GTP then binds
to the importin protein complex and this causes a conformational change such that
importin loses affinity for the cargo protein and the cargo protein is released inside
the nucleus
- step 4: the Ran GTP importin complex then diffuses back through the NPC through
the cytoplasm (because importin needs to be located in the cytoplasm)
- step 5: within the cytosol, a second protein called GTPase activating protein (GAP)
stimulates Ran GTP to hydrolyze its GTP to GDP which generates a conformational
change that causes Ran to dissociate from the importin and importin is free in the
cytosol to initiate another round of transport
- step 3B: finally, Ran GDP is returned into the nucleus via the nuclear pore complex
where another protein called guanin nucleotide exchange factor (GEF) causes release
of GDP and rebinding of GTP such that Ran GTP is not in the nucleus to facilitate the
dissociation of the cargo protein from importin; hence, the hydrolysis of Ran GTP to
Ran GDP is what provides the energy to drive nuclear protein import
- many nuclear proteins such as the histones transcription factors, DNA and RNA polymerases are all synthesized in the
cytosol and need to be imported into the nucleus via the nuclear pore complex
- GEF is predominantly located inside the nucleus so that it can maintain Ran in its GTP-bound form to promote the
dissociation of the cargo protein
- on the other hand, GAO protein is predominantly located in the cytosolic side so that it can convert Ran GTP to the GDP-
bound form and thereby promote dissociation of importin to initiate another import cycle
Nuclear Protein Export
- similar mechanism to protein important, difference is that nuclear proteins, to be
exported, must have a nuclear export signal (NES)
- step 1: a nuclear export receptor, such as exportin, binds to Ran GTP in the nucleus
which causes a conformational change in exportin such that it increases its affinity for
the nuclear export signal (NES)
- step 2: consequently, Ran GTP and exportin bind to the cargo protein via the NES,
forming a trimolecular complex
- step 3: the trimolecular complex then diffuses through the NPC into the cytosol
- step 4: in the cytosol, Ran GAP (RAN GTPase activating protein) stimulates Ran
to hydrolyze the bound GTP to GDP, shifting its conformation so that it has a low
affinity for exportin, causing Ran GDP to dissociate from exportin and the cargo
protein which causes a conformational change in exportin so that it loses affinity for
the NES and cargo protein, releasing the cargo protein into the cytosol
- step 5: after this, exportin and Ran GDP are recycled back into the nucleus via the
NPC
- in the nucleus, exportin is then ready to initiate another export cycle whereas GEFs
in the nucleus exchange the GDP for GTP to allow Ran GTP ready to induce another
transport cycle
, The GTP-binding protein Ran regulates nuclear transport
- Ran exists in two nucleotide-bound forms, called GTP-bound form and GDP-bound form
- in its GTP-bound form, it is usually active, while it is usually inactive in its GDP-bound form
- Ran has both GTPase and nucleotide exchange activity hence it is able to inherently hydrolyze its GTP to GDP and
subsequently exchange its GDP for GTP
- it is helped by two other proteins called Ran GEF or Ran GAP
- Ran GDP is converted to Ran GTP-bound form though the action of Ran GEF (Ran guanine nucleotide exchange factor)
- Rans intrinsic GTP activity and its ability to hydrolyze GTP to GDP,
releasing energy, is activated through the action of Ran GAP (Ran GTPase
activating protein)
- note how Ran has its own GAP and own GEF to facilitate the specificity
associated with nuclear transport and how either form is either localized to
the nucleus (case of GEF) or the cytosol (case of GAP)
- the difference in location leads to a high Ran GTP-GDP ratio inside the
nucleus and inversely, a low Ran GTP-GDP ratio outside the nucleus (in the
cytosol) which serves to form a concentration gradient that ultimately permits
the diffusion during nuclear transport
Co-translational targeting to ER
- this pathway is also called the secretory pathway, and in this pathway, all proteins first teach the ER and are then shuttled
to other locations such as the Golgi apparatus, plasma membrane, lysosome or secretion to the extracellular space
- for this pathway to occur, a synthesis of these proteins begins with an N-terminal sequence in the nascent protein that
directs the mRNA ribosomal protein complex to the ER membrane and subsequently initiates its translocation across the ER
membrane into the ER lumen
- finally, this signals sequence may then be cleaved off by a signal peptidase and this depends on the type of protein
(whether it is a soluble protein versus a transmembrane protein)
Co-translational targeting to the ER and transport across ER membrane for soluble proteins
- note that at the beginning that this process
specifically refers to soluble proteins
(proteins that are not membrane proteins
neither in the plasma membrane nor in the
membrane or certain organelles)
- in step 1 and step 2: once the N-terminal
signal sequence is synthesized, a signal
recognition particle (SPR) recognizes and
binds to it; SRP specifically binds to the
hydrophobic core within the N-terminal
signal sequence and binds it to its own
hydrophobic cleft in the SRP; the SRP has
an affinity for binding the N-terminal signal
sequence as well as the ribosome
- in step 3: binding of the SRP to the signal sequence causes a halt in translation and the SRP then targets the nascent
protein ribosomal complex to the ER membrane where it binds to the SRP receptor which is located in the membrane
- this brings the nascent protein ribosome into contact with the translocon (also called the Sec61 complex) which is a
membrane-bound protein that holds the translating ribosome in place and acts as a channel in the membrane through which
the polypeptide chain can be transported into the ER lumen
- this interaction with the translocon is strengthened by the binding of GTP to both the SRP and the SRP receptor
- in step 4: transfer of the nascent polypeptide ribosomal complex to the translocon stimulates the opening of the translocon
channel to admit the growing polypeptide chain adjacent to the signal sequence
- the signal sequence binds to a hydrophobic binding site within the translocon and at this point, both the SRP and the ARP
receptor then hydrolyze their bound GTP causing them to dissociate from the translocon and then are ready to initiate
another cycle of polypeptide insertion into the ER
- in step 5: as the polypeptide chain is being elongated, it passes through the translocon channel into the ER lumen and
simultaneously, a signal peptidase is then used to cleave the signal sequence and the signal sequence is rapidly degraded
- in step 6: the peptide chain continues to elongate, extruding the growing chain through the translocon into the ER lumen
- in steps 7 and 8: once translation is complete, the ribosome is released and the remained of the protein is drawn into the
ER lumen and the translocon closes; the protein is then able to continue its folding or be trafficked thereafter
