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C H A P T E R 5

PROTEIN SYNTHESIS, PROCESSING, AND TRAFFICKING

Randal J. Kaufman and Laura Popolo

The final step in the transfer of the genetic information stored in begins with the initiation codon AUG and ends with one of three
DNA into proteins is the translation of the intermediary messenger stop codons (UAA, UAG, UGA), and a 3′-untranslated region (3′
molecules, mRNAs (see Chapter 1). Protein synthesis occurs in the UTR). The 5′-end carries a 7-methylguanosine structure called a
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cytoplasm and generates a great variety of products endowed with a “cap” (m GpppN mRNA) whereas the 3′-end is polyadenylated.
wide spectrum of functions. The complete set of proteins produced These modifications are required to protect the mRNA from degrada-
by a cell is called the proteome and is responsible for the remarkable tion, for export out of the nucleus, and for efficient recruitment of
diversity in cell specialization that is typical of metazoan organisms. ribosomes for translation. Once in the cytoplasm, the 40S ribosomal
To be functional, proteins need to be properly folded, assembled, subunit binds to the cap and then scans the mRNA toward the 3′-end
often modified and transported to the final destination. The cell has until a translation start codon is encountered, usually the first AUG
in its interior several membrane-bound compartments, termed (underlined) located in a nucleotide context optimal for translation
organelles, such as the mitochondria, the peroxisomes, the nucleus initiation which is called the Kozak consensus sequence (A/
and the endoplasmic reticulum to which the proteins may be targeted. GNNAUGG). The assembly of the 60S subunit with the 40S pro-
Since each compartment serves a particular purpose, protein transport duces an 80S ribosome. A special tRNA specific for methionine,
Met
is crucial to maintain the identity and functions of each organelle. called the initiator (tRNA i ) is required for the initiation of protein
Intracellular physiology depends on the proper and coordinated synthesis at the initiation codon. Aminoacyl-tRNAs ferry amino acids
functioning of the organelles. In many cases protein folding and to the ribosome for joining together in sequence as the ribosome
processing are coupled with protein trafficking so that the targeting moves toward the 3′-end of the mRNA. The codons in the mRNA
process is unidirectional and irreversible. interact by base-pairing with the anticodon of the tRNAs so that
This chapter briefly describes how proteins are synthesized and amino acids are incorporated into the nascent polypeptide chain in
then focuses on their processing and delivery to their appropriate the right order. Translation is terminated when the ribosome encoun-
destinations within the cell. An understanding of the machines that ters a stop codon where the polypeptide is released. Typically, multiple
catalyze protein folding, assembly, processing, and targeting is relevant ribosomes are engaged in the translation of a single mRNA molecule
to the study of hematology providing a basis for an explanation of in a complex termed a polyribosome or polysome.
how malfunctions in these processes can cause hematologic Protein synthesis is divided into three phases: initiation, elonga-
disorders. tion and termination. Each phase requires a set of soluble proteins
(or factors) which transiently associate with the ribosomes and are
called initiation, elongation and termination (or release) factors that
PROTEIN SYNTHESIS are termed eIFs, eEFs, and eRFs, respectively, where the prefix “e”
indicates their eukaryotic origin. Many soluble factors required for
Among the biosynthesis of macromolecules occurring in a cell, protein synthesis belong to the G-protein (guanine nucleotide-
protein synthesis is the most important in quantitative terms. It is a binding proteins) superfamily which are regulatory molecules that
highly energy-consuming process and proceeds through a mechanism promote unidirectionality of important cellular processes such as
that has been conserved during evolution. Proteins are synthesized by hormone and growth factor signaling, membrane trafficking and
the joining of amino acids, each of which has characteristic physico- neurotransmission. Dysfunctions of G-proteins are involved in
chemical properties (see Table 5.1 for single letter designations). human diseases, including cancer.
Peptide bonds are created by the condensation of the α-carboxyl
group (COOH) of one amino acid with the α-amino group (NH 2 )
of another. The free NH 2 and COOH groups of the terminal amino REGULATION OF mRNA TRANSLATION
acids define the amino- or N-terminal end and the carboxyl- or
C-terminal end of the resulting polypeptide chain, respectively. In There are two major general regulatory steps in mRNA translation
many cases multiple polypeptide chains assemble into a functional that are mediated by the initiation factors eIF2 and eIF4. All cells
protein. For example, hemoglobin is formed by four polypeptide regulate the rate of protein synthesis through reversible covalent
chains, two α-globin chains and two β-globin chains that assemble modification of eIF2, a soluble factor required for the binding and
Met
with heme, an iron-containing prosthetic group, to yield the func- recruitment of the Met-tRNA i to the 40S ribosomal subunit.
tional protein designed to deliver molecular oxygen to all cells and eIF2 is a heterotrimeric G-protein that can exist in an inactive
tissues. form bound to GDP or an active form bound to GTP. The eIF2-
Met
The whole process of protein synthesis is orchestrated by a large GTP/Met-tRNA i ternary complex binds to the 40S subunit.
ribonucleoprotein complex, called the ribosome. The ribosome 80S Joining of the 60S subunit triggers hydrolysis of GTP to GDP and
(S stands for Svedberg unit, and refers to the rate of sedimentation) thus converts eIF2 to the inactive form whereas the opposite reaction
is typical of mammalian cells and is constituted by a large subunit of is catalyzed by a guanine nucleotide exchange factor (GEF) called
60S and a small one of 40S. Additional components are messenger eIF2B. Phosphorylation regulates eIF2 function. In reticulocytes,
RNAs (mRNAs), transfer RNAs (tRNAs), amino acids, soluble which primarily synthesize hemoglobin, heme starvation inhibits the
factors, ATP and GTP. Activation of amino acids by coupling to their synthesis of α- and β-globin chains by activating a protein kinase,
cognate tRNAs occurs before polypeptide chain initiation. This called hemin-regulated inhibitor (HRI) that specifically phosphory-
crucial function is carried out by 20 different aminoacyl-tRNA lates the α subunit of eIF2. The phosphorylated form of eIF2 binds
synthetases, one for each amino acid, which generate aminoacyl- more tightly than usual to eIF-2B, so that eIF-2B is sequestered and
tRNAs at the expense of ATP and operate a quality control on the not available for the exchange reaction. Thus, eIF2 molecules remain
coupling reaction. Eukaryotic mRNA molecules typically contain a in the GDP-bound form and translation of globin mRNA comes to
5′-untranslated region (5′ UTR), a protein coding sequence that a halt. In this manner, globin chains are not synthesized in the

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