12 - intracellular compartmeents and sorting Flashcards
three types of protein traffic
- gated transport. Through nuclear pore complexes in the nuclear envelope. Active transport of specific macromolecules and free diffusion of smaller molecules
- protein translocation. Movement of proteins across a membrane from the cytosol to a topologically distinct space. Cytosol into mitochondria, plastids, ER, peroxisomes
- vesicular transport. Transport of proteins between the lumens of two compartments via vesicles, like ER to golgi
signal sequences
sorting receptors rec them and help direct the proteins to the correct cellular compartment. Can be at the ends of the peptide or in the middle. seqs directing to the same place can have very different AA composition, but usually the properties are the same.
nuclear pore complexes (NPCs)
nuclear pores form the gates connecting cytosol and nucleus (import and export)
fully folded nuclear proteins and protein complexes can be imported which differs fundamentally from the transport of macromolecyles across the mmebranes of other organelles (via membrane-spanning transporters), which typically requires unfolding.
Major component of NPCs are nucleoporins. Nuclear localization signals (NLSs) are responsible for the selectivity of active nuclear import:
- lysine/arginine rich stretch - anywhere in protein
- NLS is rec by nuclear import proteins (importins)
- NS on one component is enough to transport entire complex.
Nuclear export signals (NES) are responible for the export of macromolecules and protein complexes. NES are rec by exportins.
small molecules can freely diffuse through the NPX but larger ones need receptor-mediated mechanisms.
nuclear import and export
Importins are soluble, cytoplasmic proteins that bind cargo (via NLS) and the nucleoporins. They move along fibrils by repeatedly binding, dissociating, and rebinding with the FG-repeat seq until inside the nucleus, where the cargo is released. Import can also require adaptor proteins
nuclear export works in reverse
Ran protein
The cell fuels the concentrating of nuclear proteins in the nucleus by harnessing energy stored in concentration gradients of the GTP-bound form of the monomeric GTPase Ran, which is required for both import and export (nucleus).
Ran is a molecular switch that can exist in two conformations, depending on whether GDP or GTP is bound. Two Ran-specific regulatory proteins: GTPase-activating protein (GAP) triggers GTP hydrolysis and converts Ran-GTP to Ran-GDP, and a nuclear guanine exchange factor (GEF) does the opposite. Ran-GAP is in cytosol, Ran-GEF in nucleus, making the cytosol full of Ran-GDP and the nucleus Ran-GTP. This gradient drives nuclear transport in the appropriate direction.
Docking of importins to FG-receptors on the cytosolic side of NPC occurs whether or not the receptors are loaded with cargo. The importins then enter the channel. If they reach the nuclear side, they are bound by Ran-GTP, which causes the receptors to release their cargo (if they have some). Ran-GDP from the cytosol does not bind to importins (or exportins), so there is no unloading of cargo in the cytosol.
Ran-GTP then guides the importin back to the cytosol, where Ran-GAP triggers hydrolyzation to Ran-GDP, which will dissociate from the receptor.
Export happens similarly, but the Ran-GTP in the nucleus promotes cargo-binding to the exportin, rather than promoting cargo dissociation in the nucleus, and opposite in cytosol.
Translocation into mitochondria
dependent on signal seq.
protein destined for mitochondria are fully synthetizes as precursor proteis in cytosol and translocated into mitochondrial lumen
TOM (translocase outer membrane) transfers proteins across the outer membrane. SAM (sorting and assembly machine) helps TOM insert certain proteins across or into the inner membrane
TIM (translocase inner membrane) compelxes transfer proteins across or into the inner membrane
OXA (cytochrome oxidase activity) inserts proteins form matrix into the inner membrane.
the precursors remain ulfoded thanks to the chaperone hsp70, which prevents it from folding before it interacts with TOM.
Stages of import:
- import receptors on TOM bind signal seq, and hsp70 is removed (removal requires ATP)
- translocation into the intermembrane space
- signal seq binds TIM, which translocates the precursor protein into the matrix
- removal of signal peptide by peptidase
end: folded mature mitochondrial matriz protein.
insertion into the inner mitochondrial membrane:
STOP transfer sequence. Cleavage of the signal peptide opens hydrophobic stretch (stop transfer seq), inducing lateral transport into the inner membrane (the protein does not enter the matrix). For an inter-membrane space protein, the protein is first inserted into the inner membrane and a protease cleaves of the stop transfer seq (hydrophobic anchor), releasing the protein.
If the protein has entered the matrix but is supposed to be in the inner membarne, OXA inserts it.
Metabolite transporter proteins are inserted into the inner membrane by TIM22 (protein does not enter matrix)
Translocation into chloroplasts
resembles mitochondrial import. Precursor proteins contain an N terminal signal seq. Uses seaprate transport processes on each membrane, requires energy.
However!
Little seq similarity between the two organelles, and no electrochemical gradient at inner membrane (only thylakoid membrane)
Import into thylakoid space: two signal seqs for sequential transport (stroma, thylakoid space), uses H+ gradient and ATP hydrolysis, four different mechanisms for transport into thylakoid spcae or thylakoid membrane (TIC = translocase inner chloroplast membrane, TOC = outer).
Transport into peroxisomes
peroxisomes contain oxidation enzymes (like catalase), is important for detoxification.
two routes of peroxisome generation: division of mature peroxisome and de novo from ER:
C-terminal signal seq on peroxisomal proteins are rec by cytosolic receptors like Pex5.
guide cargo into peroxisome lumen
multiple peroxins make u pa peroxin translocator complex - thought to be dynamic as they allow the import of folded and oligomeric proteins
ATP dependent transport.
all peroxisomal proteins are imported post-translationally
the ER
rough (synthesis, folding, fodification (like glycsylation) of proteins) and smooth (lipid synthesis and metabolism, Ca++ storage/release (like SR)
all proteins transported through the secretory pathway (soluble and membrane bound) are passing though /modified in the ER.
import is co-translational (happens while the protein is still being synthetized). the ribosome starts to make the peptide, signal seq first, which is rec by RP (signal recognition paticle), causing pause in translation. SRP binds to SRP receptor in RER membrane, ditching the ribosome and peptide there to look for a new one.
This ensures proteins like hydrolases are not released into the cytosol, and prevents the proteins from folding in the cytosol
protein translocation into the ER
co-translational (almost all, but some post translation)
SEC61 translocators open centrically and sideways. Movement through Sec61 happens when the protein is still being translated. Some proteins pass through Sec61 after full synthesis (requires BiP)
integration of membrane proteins into the ER
different types of membrane proteins:
single pass transmembrane proteins can be inserted in multiple different ways:
- N-terminal signaling seq initiates translocation, an additional hydrophobic segment in the plypep chain stops the transfer process before it has passes completely (stop-transfer signal), anchoring the protein in the membrane. the gate can open laterally to release the protein into the lipid bilayer.
- if signal seq is internal, the SRP will bind to the internal signal seq, bring the ribosome to the ER membrane, and the ER signal seq serves as a start-transfer signal that initiates translocation. After release from the translocator, the internal start-transfer seq remains in the lipid bilayer as a single membrane-spanning alpha helix.
Multipass proteins:
- combinations of start-transfer and stop-transfer signals determine the topology of multipass transmembrane proteins. An internal signal seq serves as a start-transfer signal, translocation continues until a stop-transfer signal is reached. A second start-transfer signal can then begin translocation of another part of the protein, and so on.
glycoproteins
functions of glycoproteins:
originally simple protection, in higher organisms structural changes lead to formation of mucus, which can protect lungs from pathogenic infection.
recognition function in cell-cell adhesion (selectins)
antigenic properties
regulates activity of receptors
Sifferent types of glycosylation:
- attachment of sugar groups to proteins
N-linked glycosylation is most common
N-linked glycosylation in the ER:
oligosaccharyl transferase is an ER membrane-resident protein associated with the translocon, cotranslational modification of the polypeptide and active only on lumen side.
- synthesis of standardized dolichol-linked precursor oligosaccharide
- en bloc transfer of precursor oligosaccharide to the protein by oligosaccharyl transferase
- diversity in mature glycoproteins arises from later modifications in golgi
N-linked glycosylation in ER quality control
cycles of glucosylation and deglucosylation continue until the protein reaches its native conformation, OR
the unfolded N-linked glycoprotein undergoes glucose trimming, until only the terminal glucose is left
interaction between the oligosaccharide and calnexin/calreticulin (lectins and molecular chaperones)
a glucosidase then removes the final glucose which releases the protein from calnexin/calreticulin
properly folded proteins will exit the ER, incomplete folding will lead to a GLUCOSYL TRANSFERASE adding a new glucose to the interaction with calnexin/calreticulin continues
OR
the protein is targeted for degradation. For the degradation of glycoproteins, trimming of a single mannose by the ER mannosidase in the middle of the oligosaccharide leads to an association with ER degradation-enhancing mannosidase protein EDEM).
the protein is then exported from the ER via retrotranslocation, and are deglycosylated and ubiquitylated (targets them for proteasome degradation)
Misfolded proteins in the ER can send signals to the nucleus
- msisfolded proteins in ER signal the need for more ER chaperones. They bind to and activate a transmembrane kinase
- transmembrane kinase will unmask an endoribonuclease activity
- endoribonuclease cuts specific RNA molecules at two positions, removing an intron
- two expns are ligated to form an active mRNA
- mRNA is translated to make a transcription regulator
- transcription regulator enters nucleus and activates genes encoding ER chaperones
- ER chaperones are made in ER, where they help fold proteins.
