3.2.3 Transport of Newly Synthesized Proteins to Their Correct Destinations in the Cell
83
and co-eIF-2B; the phosphorylation of the elF2a subunit seen at the beginning of development
appears to have no influence on the formation of
the initiation complex [295].
3.2.3 'fiansport of Newly Synthesized
Proteins to Their Correct Destinations
in the Cell
After their synthesis on free polysomes or on the
ribosomes bound to the rough endoplasmic reticulum, the polypeptides must be transported to
their particular destinations in the cell: secretory
proteins are transported out of the cell by exocytosis; integral membrane proteins are built into
the plasma membrane; and the proteins of the
nucleus, mitochondria, microbodies (peroxisomes, glyoxysomes, glycosomes) and lysosomes
are introduced into their respective organelles.
During their translocation, proteins must normally pass at least one cellular membrane. The
signals for the distribution to different cell compartments are contained in the amino acid
sequence of the primary translation products
[21, 90, 262]. Identification of these signals is
made possible by genetic defects that disturb the
translocation of secretory, lysosomal or membrane proteins. Proteins without an address
remain in the cytoplasm; proteins linked experimentally with a translocation signal are transported into the relevant compartment [20]. During or after translocation, the polypeptides may
be changed by partial proteolysis, substitution or
conjugation, linked to non-protein components
or built into supra-molecular structures. Proteolytic processes can bring about the removal of the
N-terminal methionine or of the terminal translocation signals, or the maturation (processing) of
pro-enzymes, blood-clotting factors, complement
components or hormone precursors. Substitutions and conjugations create the numerous
altered protein components described previously.
All these processes will be dealt with in the sections on the relevant proteins. Unfortunately,
there are very few data on the comparative biochemistry of translocation and post-translational
modifications.
The signals and mechanisms of translocation
appear, in principle, to be the same in all organisms. Hence, polypeptides encoded by foreign
genetic material are correctly distributed in Xenopus laevis oocytes, and even in E. coli. After
injection of the corresponding mRNA into oocytes, typical secretory proteins, like ovalbumin and
other egg-white proteins, immunoglobulins or
insect vitellin, are released into the medium; rabbit haemoglobin is retained in the cytoplasm; and
cytochrome P-450 is introduced into membranes
[147]. Firefly luciferase, synthesized in the cells of
various insects and mammals, and even of higher
plants and yeast, is translocated correctly to the
peroxisomes [86]. The signals are so conserved in
evolution that the normally cytoplasmic a-globin
chain is taken up by the endoplasmic reticulum if
it is coupled to the signal of the bacterial ~lactamase [20]. The fact that human genes are not
only transcribed and translated in bacteria but, in
many cases, also correctly processed is made use
of in industry. Post-translational modification is
also uniform within very wide limits. Thus, after
injection of the mRNA for phaseolin into Xenopus oocytes, this plant storage protein is not only
correctly translated but also glycosylated [173].
There are, however, species- and cell-specific
mechanisms for the further processing of newly
synthesized polypeptides; after the introduction
of the rat prepro-insulin gene into E. coli, proinsulin, and not insulin, is released into the
medium [147]. Likewise, injection of the mRNA
for the bee toxin prepro-melittin into Xenopus
oocytes does not result in the release of melittin,
but pro-melittin is stored for many weeks in cellular vesicles; here, in contrast to the situation in
the poison glands, the C-terminal glycine is not
removed and the export of the protein is blocked
[149].
Secretory proteins find their way out of the
cytoplasm via the lumen of the endoplasmic reticulum (ER) and the Golgi apparatus into specific
transport vesicles, in which they are then transported to the cell surface. Because the vesicles of
the Golgi apparatus and those of the ER arise by
budding the proteins must only cross one membrane, that of the ER. According to the signal
hypothesis, an N-terminal, non-polar signal
sequence is responsible and this is recognized by a
signal recognition particle (SRP) [21, 90]. Polypeptide synthesis begins on free, cytoplasmic
polysomes. Through binding of an SRP to the
translation complex, the elongation of the polypeptide is stopped at a length of about 70 amino
acids (Fig. 3.3). The complex of SRP, ribosome
and incomplete polypeptide chain binds to an
SRP receptor on the ER membrane (a "docking"
protein), and the SRP is split off. Now the signal
sequence can interact with the signal sequence
receptor and elongation is restarted. The SRP has
three different tasks to accomplish (signal
recognition, translation inhibition, and interac-
83
and co-eIF-2B; the phosphorylation of the elF2a subunit seen at the beginning of development
appears to have no influence on the formation of
the initiation complex [295].
3.2.3 'fiansport of Newly Synthesized
Proteins to Their Correct Destinations
in the Cell
After their synthesis on free polysomes or on the
ribosomes bound to the rough endoplasmic reticulum, the polypeptides must be transported to
their particular destinations in the cell: secretory
proteins are transported out of the cell by exocytosis; integral membrane proteins are built into
the plasma membrane; and the proteins of the
nucleus, mitochondria, microbodies (peroxisomes, glyoxysomes, glycosomes) and lysosomes
are introduced into their respective organelles.
During their translocation, proteins must normally pass at least one cellular membrane. The
signals for the distribution to different cell compartments are contained in the amino acid
sequence of the primary translation products
[21, 90, 262]. Identification of these signals is
made possible by genetic defects that disturb the
translocation of secretory, lysosomal or membrane proteins. Proteins without an address
remain in the cytoplasm; proteins linked experimentally with a translocation signal are transported into the relevant compartment [20]. During or after translocation, the polypeptides may
be changed by partial proteolysis, substitution or
conjugation, linked to non-protein components
or built into supra-molecular structures. Proteolytic processes can bring about the removal of the
N-terminal methionine or of the terminal translocation signals, or the maturation (processing) of
pro-enzymes, blood-clotting factors, complement
components or hormone precursors. Substitutions and conjugations create the numerous
altered protein components described previously.
All these processes will be dealt with in the sections on the relevant proteins. Unfortunately,
there are very few data on the comparative biochemistry of translocation and post-translational
modifications.
The signals and mechanisms of translocation
appear, in principle, to be the same in all organisms. Hence, polypeptides encoded by foreign
genetic material are correctly distributed in Xenopus laevis oocytes, and even in E. coli. After
injection of the corresponding mRNA into oocytes, typical secretory proteins, like ovalbumin and
other egg-white proteins, immunoglobulins or
insect vitellin, are released into the medium; rabbit haemoglobin is retained in the cytoplasm; and
cytochrome P-450 is introduced into membranes
[147]. Firefly luciferase, synthesized in the cells of
various insects and mammals, and even of higher
plants and yeast, is translocated correctly to the
peroxisomes [86]. The signals are so conserved in
evolution that the normally cytoplasmic a-globin
chain is taken up by the endoplasmic reticulum if
it is coupled to the signal of the bacterial ~lactamase [20]. The fact that human genes are not
only transcribed and translated in bacteria but, in
many cases, also correctly processed is made use
of in industry. Post-translational modification is
also uniform within very wide limits. Thus, after
injection of the mRNA for phaseolin into Xenopus oocytes, this plant storage protein is not only
correctly translated but also glycosylated [173].
There are, however, species- and cell-specific
mechanisms for the further processing of newly
synthesized polypeptides; after the introduction
of the rat prepro-insulin gene into E. coli, proinsulin, and not insulin, is released into the
medium [147]. Likewise, injection of the mRNA
for the bee toxin prepro-melittin into Xenopus
oocytes does not result in the release of melittin,
but pro-melittin is stored for many weeks in cellular vesicles; here, in contrast to the situation in
the poison glands, the C-terminal glycine is not
removed and the export of the protein is blocked
[149].
Secretory proteins find their way out of the
cytoplasm via the lumen of the endoplasmic reticulum (ER) and the Golgi apparatus into specific
transport vesicles, in which they are then transported to the cell surface. Because the vesicles of
the Golgi apparatus and those of the ER arise by
budding the proteins must only cross one membrane, that of the ER. According to the signal
hypothesis, an N-terminal, non-polar signal
sequence is responsible and this is recognized by a
signal recognition particle (SRP) [21, 90]. Polypeptide synthesis begins on free, cytoplasmic
polysomes. Through binding of an SRP to the
translation complex, the elongation of the polypeptide is stopped at a length of about 70 amino
acids (Fig. 3.3). The complex of SRP, ribosome
and incomplete polypeptide chain binds to an
SRP receptor on the ER membrane (a "docking"
protein), and the SRP is split off. Now the signal
sequence can interact with the signal sequence
receptor and elongation is restarted. The SRP has
three different tasks to accomplish (signal
recognition, translation inhibition, and interac-