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48 Part I Molecular and Cellular Basis of Hematology

processing of propolypeptides into their mature forms occurs after 5.1). The first sorting decision occurs after approximately 30 amino
the Arg-Xxx-Lys-Arg motif and is mediated by the general protein acids of the nascent polypeptide are extruded from the ribosome. If
processing protease FURIN. FURIN is expressed in all cell types the nascent polypeptide lacks a “signal sequence,” most often found
and removes propeptides from many proteins, including clotting near the amino-terminal end, the translation of the polypeptide is
factors, growth factors, and proteases, and is responsible to convert completed in the cytosol. Then the protein either remains in the
these proteins into their functional forms. Other members of the cytosol or is posttranslationally incorporated into one of the indicated
proprotein convertase (PC) family cleave after pairs of dibasic organelles (see Fig. 5.1). If the protein does contain an amino-terminal
amino acids (for example Lys-Arg) and are expressed in specific signal sequence it is imported co- or posttranslationally into the ER
cell types, such as neuroendocrine cells. The PCs are responsible or mitochondrion. Trafficking of proteins from the ER to the Golgi
for cleavages that occur to generate many hormones, such as compartment and lysosomes occurs via vesicle budding and fusion
insulin, glucagon, and adrenocorticotropic hormone. events (see Fig. 5.1).
Other modifications such as N-glycosylation, O-glycosylation and
attachment of a glycosylphosphatidylinositol (GPI) anchor will be Targeting of Nuclear Proteins
discussed in a separate section.
Finally, proteins can also be susceptible to nonenzymatic modifi- One of the distinctive features of all eukaryotic cells is that the
cations that take place under particular conditions, such as during genome is contained in an intracellular compartment called the
oxidative stress or in the presence of excess glucose in the blood as in nucleus. The nucleus is bounded by a double membrane that forms
the case of hemoglobin glycation. the nuclear envelope (NE) (see Fig. 5.1). The outer nuclear membrane
is continuous with the ER and has a polypeptide composition distinct
from that of the inner membrane. About 3000 nuclear pore com-
PROTEIN DEGRADATION plexes (NPCs) perforate the NE in animal cells. Although NPCs
allow unrestricted, bidirectional movement of molecules smaller than
The level of a protein in the cell results from the balance between its 40,000 Daltons, traversal of larger molecules across NPCs is tightly
synthesis and degradation and a protein’s half-life can vary from regulated. NPCs are approximately 120 nm in external diameter and
minutes to hours. Moreover, the cell eliminates proteins that contain comprise approximately 50 different proteins (nucleoporins),
mutations that cause protein misfolding. Such misfolded proteins are arranged in a complex cylindrical structure with an octagonal sym-
marked for destruction and then degraded to avoid toxic effects of metry. Nucleoporins constitute the scaffold of the NPC and are
their accumulation. The breakdown of these molecules is achieved in arranged in rings. In the inner ring, nucleoporins containing repeats
two major phases. First, the molecules are tagged with ubiquitin, of two hydrophobic amino acids, phenylalanine and glycine
which is covalently linked to the substrate protein as described earlier. (FG-repeats), seem to be essential for the movement of the cargo-
Second, the tagged molecules are ferried to an ATP-dependent pro- carrier complexes and for creating a selectivity barrier against the
tease complex called the 26S proteasome, a multi-subunit molecular diffusion of nonnuclear proteins. The FG-nucleoporin filaments
machinery specialized in protein destruction. Peptides and amino protrude toward the inner core of the NPC and the weak hydrophobic
acids derived from protein disposal are recycled. interactions between the FG-repeats and the cargo-carrier complexes
Since its first discovery in carrying out the disposal of damaged mediate the passage of molecules.
and misfolded proteins, protein ubiquitylation was found associated NPCs are capable of importing and exporting molecules or
to an increasing number of specific regulatory events involving a complexes, provided that the molecules have an exposed nuclear
selective degradation of key regulatory proteins. Thus, ubiquitylation localization signal (NLS) or nuclear export signal (NES). These
is responsible for regulating a wide array of cellular processes includ- signals are not always easy to predict. In Table 5.1 some of the
ing differentiation, tissue development, induction of inflammatory best-known signals are listed. The function of these signals in import-
responses, antigen presentation, cell cycle progression and pro- ing or exporting a protein was analyzed by critically testing both the
grammed cell death also named apoptosis (Chapter 18 will review effects of amino acid substitutions on transport and the capability of
cell death). In addition, according to the pattern of modification of the signal to target in or out of the nucleus an attached reporter
the target proteins (monoubiquitination, polyubiquitination) differ- protein. The nuclear localization signals are not cleaved off as occurs
ent proteins can trigger DNA repair (monoubiquitination of for other signals (see later) and thus can function repetitively. Can-
N-terminal tails in histones) or be subjected to endocytosis (monou- didates exposing signals for nuclear import (i.e., transcription factors,
biquitination of surface receptors). coactivators or corepressors, DNA repair enzymes, ribosomal proteins
and mRNA processing factors, etc.) or export (ribosomal subunits,
SORTING FROM THE CYTOSOL INTO OTHER mRNA-containing particles, tRNAs, etc.) are transported through
the NPC in association with soluble carrier proteins, called karyo-
COMPARTMENTS pherins (also called importins, exportins or transportins), which
function as shuttling receptors for different protein cargos. According
Most proteins are synthesized on free polysomes and remain in the to the direction of transport, they are divided into two groups: (i)
cytosol. These include enzymes involved in many metabolic and importins, if they bind their cargo on the cytoplasmic side of the
signal transduction pathways, proteins required for mRNA transla- NPC and release it on the other and (ii) exportins if they bind the
tion or assembly of the cytoskeleton. Other proteins are imported cargo in the nucleus and release it in the cytoplasm.
from the cytosol into organelles including the nucleus, the mitochon- Ran is a small Ras-like GTPase, belonging to the G-protein
drion, and the peroxisome (Fig. 5.1). superfamily, that controls both the docking of carrier proteins with
In general, there are two types of protein trafficking. In one type, their cargo and the directionality of transport through cycles of GTP
the protein crosses a lipid bilayer. The polypeptide crosses the mem- binding and hydrolysis. Fig. 5.2 exemplifies a cycle of import into the
brane in an unfolded state through an aqueous channel composed of nucleus. An importin binds the cargo in the cytosol and then associ-
proteins. In the second type, the protein does not traffic across a lipid ates with Ran-GDP for trafficking into the nucleus where a Ran-GEF
bilayer and is exemplified by trafficking into the nucleus or from the (guanine-nucleotide exchange factor) catalyzes GTP exchange for
endoplasmic reticulum (ER) to the Golgi compartment. In these GDP on Ran that triggers cargo release. The importin-Ran-GTP
cases, proteins and protein complexes are transported in their folded/ complex is transported back to the cytoplasm where the conversion of
assembled state. GTP to GDP is stimulated by a Ran-GAP protein (GTPase-activating
The trafficking events are governed by sorting signals (i.e., short protein) that causes dissociation of Ran from the importin which
linear sequences or three-dimensional patches of particular amino can initiate a new cycle. The movement of cargo from the nucleus
acids) and by their cognate receptors (see some examples in Table to the cytoplasm occurs by formation of a Ran-GTP-exportin-cargo