Fall 2017

11. PROTEIN SORTING I: NUCLEUS AND MITOCHONDRION

1. PROTEIN SORTING
Ø Sorting refers to the movement to appropriate destinations
Ø Eukaryotic cells are compartmentalized
Ø Each organelle requires a specific protein (enzymes, transporters) to function
Ø Most of these are synthesized somewhere in the cytosol and must be
transported (sorted) to appropriate sites
• Note: This a spontaneous process and not an anthropomorphic
process

2. MOVEMENT OF PROTEINS BETWEEN COMPARTMENTS

I. Gated Transport: Nucleus
Proteins move between cytosol and nucleus through nuclear pore
complexes in the nuclear envelope

II. Transmembrane Transport: Mitochondria & ER

Transmembrane protein translocators directly transport specific proteins
across a membrane from the cytosol into a space topologically distinct.
Must remain unfolded and snake through translocators.
Integral membrane protein uses the same translocators but translocate
partially to be embedded within bilipid layer

III. Vesicular Transport: Secretory Pathway

Membrane-enclosed transport intermediates-which may be small,
spherical transport vesicles or larger, irregularly shaped organelle
fragments-ferry proteins from one
compartment to another. The transport
vesicles and fragments become loaded
with a cargo of molecules derived from the
lumen of one compartment as they bud
and pinch off from its membrane; they
discharge their cargo into a second
compartment by fusing with the membrane
enclosing that compartment

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3. SIGNAL SEQUENCE OR PATCH

(A) Signal Sequence
Signal that is composed of consecutive amino acid sequence at the terminal end
of protein
(B) Signal Patch
Signal amino acids are located are the internal part of protein (signal sequence is
spread then when they are folded forms the signal patch)

The synthesis of all proteins begins on ribosomes in the cytosol, except for the
few that are synthesized on the ribosomes of mitochondria and plastids. Then,
their fate depends on their amino acid sequence, which can contain sorting
signals that direct their delivery to locations outside the cytosol

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CYTOSOL & NUCLEUS

4. PROTEIN SORTING BETWEEN CYTOSOL & NUCLEUS
• Important proteins (polymerases, GRP) are synthesized in the cytosol and
exported in the nucleus
• Other macromolecules are synthesized in the nucleus and exported to the
cytosol
• Bidirectional Traffic
• Nuclear envelope consists of a nuclear pore complex that is selective in
the compartmentalization of proteins.

Nuclear Pore Complex

• Perforated nuclear envelope
• Octagonal structure
• Composed of nucleoporins
o Ring, Channel & Scaffold
• Selectively allow passages of molecules

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• Structure consists an aqueous pore

• Small proteins traverse by passive diffusion
• Recent evidence indicates a tangled meshwork lines to block passive
diffusion of large molecule (maintain thermodynamic stability)
• Large molecule à active transport mechanism
• Binding energy cease out the meshwork
• Proteins travel in a folded conformation

Nuclear Import Receptors (NIR)

• NIR binds to nucleoporins and nuclear localization signals (NLS)
• NLSs are signal sequences or patches
• NLS-receptor recognition initiates import of cargo proteins
• Sometimes adaptor proteins involved
• Export works the same way, but in reverse, & uses Nuclear Export
Receptor (NER)

Directional Transport
• Important and export consumes energy
• A GTPase called Ran acts as a molecular switch
• Directionality occurs primarily because Ran-GDP is concentrated in the
cytosol, Rand-GTP is concentrated in the nucleus

• This gradient of Ran-GTP/GDP drives nuclear transport in

appropriate direction*

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Compartmentalization of Ran-GDP & Ran-GTP

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• GTPase-Activating Protein (GAP) hydrolyzes GTP, converting Ran-GTP

to Ran-GDP
• Guanosine Exchange Factor (GEF) promotes GDP-GTP exchange,
converting Ran-GDP to Ran-GTP
• The activity of GAP and GEF maintain the Ran-GDP/GTP gradient that
drives import and export

CYTOSOL & MITOCHONDRION

5. PROTEIN SORTING TO THE MITOCHONDRION
• Most proteins are encoded in the nucleus and must be imported to the
mitochondria
• Post-translational translocation
• Proteins are transported to matrix or inserted into membrane
• Mitochondrial proteins are first fully synthesized as mitochondrial
precursor proteins in the cytosol and then translocated into mitochondria
by post-translational mechanism

The Mitochondrial Signal Sequence

• Exposed amino acid sequence that directs protein to appropriate
address
• Mitochondrial signal sequence is modified as an alpha-helix
• (+) charged amino acids are exposed at one
side, uncharged ones at the other end

Protein Translocator Complex

• TOM: imports all mito-destined proteins to intermembrane space;
membrane insertion at outer membrane
• TIM: imports proteins to matrix (TIM 23); insertion into inner membrane
(TIM 23); insertion of carrier proteins (TIM 22)
• OXA Complex: insertion of proteins into IMS

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Translocation Across the OM

Formation of precursor protein

1. Cytosolic Hsp70 Chaperones (Interacting Proteins) à Maintain unfolded

structure
2. Binding and recognition of signal sequence at TOM receptor proteins (TOM
20, TOM 5)
3. Transfer of signal sequence and polypeptide to TOM channel (TOM 40)
4. Molecular chaperones stripped away by ATP hydrolysis
5. Unfolded polypeptide fed through the TOM channel

Translocation Across the IM

6. Polypeptide bound to TIM complex
7. Electrochemical H+ gradient pulls positively charged signal sequence through
TIM
8. Signal sequence cleaved away by Matrix Processing Peptidase (MPP)

CROSS-BRIDGE RATCHET MODEL

9. Mitochondrial Hsp70 associates with the polypeptide chain
10. ATP hydrolysis drives conformational change in Hsp70 that actively pulls
chain through

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Insertion into the Inner Membrane (Integral Protein)

11. When SS1 reaches the matrix
(A) SS2 binds to TIM23, SS1 is cleaved, insertion in the IM by TIM23
(B) SS1 is cleaved, SS2 guides chain to OXA, insertion in the IM by OXA