Download 1 Protein Secretion: Targeting to the ER I. Introduction nucleus ER

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Protein Secretion: Targeting to the ER
I. Introduction
nucleus
plasma
membrane
ATP
ER
ATP
ATP
lysosome
ATP
ATP
ATP
Golgi
ATP
mitochondria
secretory vesicle
Proteins are made in the cytoplasm, but many of them must end up in various
organelles in the cell, such as the ER, the Golgi, mitochondria or chloroplasts,
secretory vesicles, and lysosomes (vesicles containing enzymes that degrade
proteins)(see illustration in MBOC p. 552 Fig. 12-1). In addition, proteins are often
found outside the cell; for example, 7g per 100 mL of blood is the protein albumin.
Many proteins are also stuck in the plasma membrane in all sorts of ways - with the
N-terminus in the cytoplasm and the C-terminus on the outside, vice versa, with
both ends either in the cytoplasm or on the outside, with several domains crossing
the membrane ending with the ends on opposite sides, or other arrangements (see
MBOC p. 486 Fig. 10-13 for illustration). How do proteins get to all these places?
II. Ribosomes attached to ER
With the invention of the electron microscope, the resolution at which cells could be
observed improved, since the wavelength of visible light is about 500 nm while that
of an electron is about an angstrom. As part of his efforts to figure out the
mechanism of protein secretion, George Palade used this technique to look at
pancreas cells (which specialize in secreting digestive enzymes) and saw that each cell
was full of little blobs, most of them attached to vesicle membranes and a few free in
the cytoplasm (for electron micrograph, see V&V p. 578, Fig. 12-31). He identified
these blobs as ribosomes.
A subsequent experiment verified that these secretory cells have ribosomes attached
to ER membranes. In this experiment, the cells were gently homogenized and
fractionated on a sucrose density gradient, which separated the components such that
the heaviest ones were at the bottom and the lightest ones at the top. The plasma
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membranes appeared near the top, at about 30% sucrose. Below that were vesicles
without blobs, and below them (heavier) were vesicles with blobs. The vesicles
without blobs turned out to be light (ribosome-free) ER, while the vesicles with blobs
were heavy ER with ribosomes attached (see illustration in MBOC p. 581 Fig. 12-37
and below).
light
plasma
membrane (30% sucrose)
vesicles w/ no ribosomes
vesicles w/ ribosomes
heavy
If a vesicle from the ER fuses with the plasma membrane, it forms a continuous
surface, so that what was previously inside the ER or vesicle (as opposed to in the
cytoplasm) is outside the cell (see illustration in MBOC p. 555 Fig. 12-4). As a result,
getting a protein into the ER is a big first step in secreting it. Since proteins are
synthesized by ribosomes, targeting a ribosome to the ER is a step toward getting the
protein it synthesizes into the ER and ultimately out of the cell. But what directs
certain ribosomes to ER membranes?
III. Signal hypothesis
When Cesar Milstein isolated RNA from MOPC 21 myeloma cells and carried out an
in vitro translation using free ribosomes and radioactive amino acids, he found that
the in vitro synthesized protein for the light chain of IgG1 immunoglobulins was a
little bit bigger (and ran higher on an SDS-PAGE gel) than the protein isolated from
the secreted immunoglobulin. He figured the in vitro synthesized protein must
have an extra set of amino acids at either the N- or the C-terminus, and protein
sequencing revealed that it was at the N-terminus. This finding suggested that newly
synthesized proteins destined for secretion have a transient N-terminal signal
sequence that targets them for secretion.
In 1975 Blobel, a grad student of Palade, proposed this idea as the signal hypothesis.
To prove his hypothesis he took all the mRNA from pancreas cells, translated these
mRNA in vitro, and compared the sequences of the synthesized proteins with those
of the corresponding mature proteins. He found that almost none of the N-terminal
sequences before the seventeenth to twenty-fourth amino acid in the synthesized
proteins were part of the mature sequences, and the N-terminal regions all contained
a significant number of hydrophobic amino acids. The initial sequence was in fact a
signal sequence that directs proteins destined to leave the cytoplasm to the ER. Other
signals direct proteins to other organelles. Thus signal sequences work like zip codes
in directing their proteins to different regions in or out of the cell. (See MBOC p. 558
Fig. 12-8 and Table 12-3.) The signal sequence of a nascent polypeptide consists of
positively charged amino acids (usually Lys or Arg) at the N-terminus, followed by a
minimum of seven hydrophobic residues, and finally a stretch of random amino
acids containing small aliphatic amino acids at positions -1 and -3 relative (first and
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third position away from the start of the mature sequence). (see V&V p 309 Fig. 1143).
As a background experiment to determine whether a translated protein gets into the
ER, preprolactin was synthesized in vitro and then exposed to proteases in the
presence or absence of microsomes (vesicles produced from self-closing ER
fragments). In the absence of vesicles the proteases chewed up the protein so that it
could not be found on a gel, while in the presence of vesicles the protein remained
intact, indicating that it had entered the vesicles and was protected from the
proteases. (See MBOC p. 559 Panel 12-1, upper right.)
So how does a signal sequence get a protein into the ER?
IV. Signal recognition particle (SRP)
When Peter Walter extracted the rough (ribosome-loaded) ER with salt, he was able
to isolate a fraction that was essential for protein entry into vesicles. The fraction
contained an organized complex now called the signal recognition particle (SRP)
which is composed of a 300-base 7S RNA and six polypeptides. He found that if he
added the SRP during in vitro translation, translation stopped. If he then added ER
vesicles, he could relieve the arrest.
SRP 54, one of the polypeptides in SRP, was found to bind directly to the signal
sequence. This binding was supported by the finding that adding a short synthetic
signal sequence could compete with the nascent protein's signal sequence for SRP 54
binding and thus block the arrest of translation. SRP 54 is composed of three
domains, including an N-terminal GTPase. The GTPase domain acts as a timing
device based on the fact that GDP remains stuck for some time after hydrolysis. The
M domain form a hydrophobic side of a helix which recognizes and binds the
hydrophobic signal sequence.
The SRP acts as a proofreading device; it only binds if the signal sequence has charged
groups at the N-terminus, followed by six or seven hydrophobic residues, and finally
small amino acids. When the SRP contacts both the signal sequence and the
ribosome, GDP is released, GTP binds, and the GTP-bound form then binds tightly to
the ribosome, where it arrests translation and stays attached.
Why do vesicles relieve the arrest of translation?
V. SRP receptor (SR)
By gently proteolyzing vesicles and looking for a gel band which changed size,
Dobberstein and his group found a complex which they called the S RP receptor (SR).
The SR is composed of two polypeptides, α and β. The β-chain crosses the
membrane, and both chains contain GTPase domains.
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α
β
N
N
Only GTP-bound SR can interact with GTP-SRP; when they contact each other the
SRP releases the signal sequence, and both hydrolyze their GTP to GDP.
VII. Summary - Requirements for a protein to get out of the cytoplasm are:
1) signal sequence for targeting
2) SRP (in animal cells), present in the cytoplasm
3) SR at the ER membrane to transfer the signal sequence to Sec 61p
4) Sec 61p to form a channel specific for protein translocation, which will be discussed
in the next lecture.