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50 Part I Molecular and Cellular Basis of Hematology

Ran • GDP Ran • GTP Importin Cargo and assembly machinery (MIA) that drives the import of proteins
with cysteine-rich motifs, the sorting/assembly machinery (SAM)
G G that inserts precursors of proteins with a transmembrane β-barrel
D T domains in the MOM and the mitochondrial import machinery
P P NLS
(MIM) that import outer membrane proteins with α-helical trans-
membrane segments after their recognition by the Tom70 subunit of
+ G the TOM complex. 2
T G T In the context of cell biology, mitochondria play relevant roles in
P P
GEF GDP apoptosis, in the communication with the ER and in oxidative stress.
G GTP Among the proteins associated with the cytosolic side of MOM, those
D of the Bcl-2 family have both pro- or antiapoptotic functions. In
P
Nucleoplasm NPC addition, recent studies unveiled an ER-mitochondria linkage that is
2+
important in Ca homeostasis and phospholipid biogenesis whereas
oxidative stress generated in the mitochondria is connected to cell
aging and senescence.
Cytoplasm
Targeting of Peroxisomal Proteins
P i H 2 0
Cargo
complex G Peroxisomes are membrane-bound compartments in which oxidative
D GAP G
P T reactions that generate hydrogen peroxide, such as β-oxidation of
P fatty acids, occur. In this organelle hydrogen peroxide is rapidly
+ degraded by catalase to prevent oxidative reactions that have potential
damaging effects on cellular structures. A single membrane surrounds
the peroxisome that encloses an interior matrix. This organelle lacks
a genetic system and transcriptional/translational machinery. There-
fore, all peroxisomal proteins are nuclear-encoded. Their mRNAs are
Fig. 5.2 MECHANISM OF PROTEIN IMPORT INTO THE NUCLEUS. translated on cytosolic-free ribosomes and then proteins are imported
The figure shows the different localization of Ran-GAP and Ran-GEF that posttranslationally by proteins called peroxins (Pex). 3
regulate the direction of nuclear transport. A Ran-GTP-exportin-cargo The targeting of matrix proteins is directed by two types of per-
complex is transported from the nucleus to the cytoplasm where a Ran-GAP oxisomal targeting signals (PTSs). Type 1 is a carboxyl-terminal tri- or
stimulates the GTPase activity of Ran. Ran-GDP dissociates from the tetra-peptide (PTS1) whereas type 2 is an amino-terminal peptide of
complex and induces the release of the cargo from exportin. Ran-GDP is nine amino acids (PTS2) (see Table 5.1). Two cytosolic Pex, Pex5 and
transported back to the nucleus by association with an importin-cargo Pex7, recognize PTS1 and PTS2. Pex5p is sufficient for targeting
complex and in the nucleus the activity of a Ran-GEF triggers GDP-GTP- PTS1-containing proteins whereas both Pex5 and Pex7 are required
exchange and this induces the dissociation of the cargo from importin. for import of PTS2-containing proteins. These proteins function as
Ran-GTP binds an exportin-cargo complex and another cycle begins. GAP, cargo receptors; they bind cargo proteins in the cytosol, release them
GTPase-activating protein; GDP, guanosine diphosphate; GEF, guanine into the matrix and cycle back to the cytosol. In humans, Pex5 has
nucleotide exchange factor; GTP, guanosine triphosphate; NLS, nuclear two isoforms, Pex5S and Pex5L. The latter has an additional exon
localization signal, NPC, nuclear pore complex. (Adapted from Lodish H, Berk that is required for binding of Pex7 to Pex5. Matrix proteins cross
A, Matsudaira P, et al: Molecular cell biology, ed 5, New York, 2003, W.H. the membrane in a folded state, or even as oligomers. Other peroxins
Freeman.) are involved in the import of peroxisomal membrane proteins
(PMPs). Pex3, Pex16 and Pex19 are thought to be involved in the
formation of the peroxisomal membrane and/or insertion of PMPs
in the peroxisomal membrane. Experimental evidence suggests the
existence of two routes by which PMPs can reach peroxisomes, one
Precursor proteins usually have one of two targeting signals: (1) direct route and one via the ER. Some PMPs are targeted to peroxi-
an amino-terminal presequence that is generally between 10 and 80 somes by Pex19 and others are not. The PMP binds Pex19,
amino acids long and forms an amphipathic α-helix, which is rich in a soluble recycling receptor/chaperone, to form a cargo-receptor
positively charged, hydrophobic and hydroxylated amino acids (Table complex that docks on Pex3 in the peroxisomal membrane and then
5.1); or (2) a less well-defined, hydrophobic targeting sequence dis- membrane insertion occurs. The indirect targeting via the ER involves
tributed throughout the protein. The translocase of the outer mem- only some PMPs, Pex3 for example, and so far, has been demonstrated
brane (TOM) complex functions as a single entry point of incoming only in yeast where the Sec61 translocon is required. Other Pex
precursors into the mitochondria and is crucial for the biogenesis of proteins are involved in ubiquitination. Ubiquitination of the recep-
the organelle and for the viability of eukaryotic cells. Preproteins tors has been proposed to function in concert with ATPases associated
+
translocate through the TOM complex in an N-to-C direction in an to diverse activities (AAA ATPases) that move proteins across mem-
unfolded state. TOM translocase is a heteromolecular protein brane using an ATP-dependent mechanism that resembles the ret-
complex whose central component is Tom40, an essential protein that rotranslocation of misfolded proteins from ER lumen to the cytosol
forms the protein-conducting channel. After crossing the outer and in recycling the receptors to the membrane surface.
membrane, proteins segregate according to their signals and recognize One consequence of the existence of two different mechanisms
two distinct translocases of the inner membrane (TIM23 and for protein import is that when the import of matrix proteins is
TIM22). Presequence-containing proteins are directed to the TIM23 defective, membrane ghosts of peroxisomes persist in the cells. In
complex that mediates transport across the inner membrane, a process contrast, when the import of membrane proteins is impaired, neither
which requires the electrochemical membrane potential and the ATP- normal peroxisomes nor membrane ghosts are present. Defects in
driven action of the matrix heat shock protein 70 (mtHsp70). Once PEX3 underlie Zellweger syndrome that is characterized by the pres-
in the matrix, the presequence is often cleaved by a mitochondrial ence of empty peroxisomes and abnormalities in the brain, liver, and
processing peptidase (MPP). Proteins with internal targeting signals kidney that cause death shortly after birth. Other syndromes in
are guided to the TIM22 complex. Membrane insertion at the TIM22 peroxisome biogenesis were identified and their occurrence underlines
complex is also dependent on the membrane potential. Other the essential role of these organelles for human metabolism and
machineries of protein sorting are the intermembrane space import development. 4