Central Dogma and Endomembrane System
Getting Glucose Transporter out to the membrane – eukaryotic
When insulin binds to the insulin receptor on a liver cell a signal is sent in to the nucleus
and the response is to activate the two genes for glucose transporter protein, a membrane
protein that will allow glucose to then diffuse into the cell. Within the nucleus of the
eukaryotic cell exists the chromatin, which is a collection of all the chromosomes (46 in
humans). A eukaryotic chromosome is a linear piece of DNA plus all of the proteins
associated with it including the histones, which serve to organize the chromosomes.
Chromosomes are analogous to books as they contain instructions to build every RNA and
polypeptide/protein molecule in the cell. This would make the nucleus like the library of
the cell. The short sections of the chromosomes that contain this information are known as
genes and they make up approximately one percent of the genetic material. Genes might be
thought of as individual pages of the book. All genes contain instructions to first build RNA.
There are three main types of genes – mRNA, rRNA and tRNA genes. The enzyme that is
able to read the genes and build the complementary RNA molecules is called RNA
polymerase. This process is known as transcription because the genes are being essentially
rewritten in the same “language” since RNA is very similar to DNA as they are both nucleic
acids. The rRNA genes are located in the nucleolus of the nucleus, which is where
ribosomes are formed. The rRNA or ribosomal RNA made by RNA polymerase in this region
will fold up and form 65% of the ribosome with the other 35% being a number of proteins
imported from the cytosol. The ribosome is actually made in two distinct parts known as
the small and large subunits. The tRNA genes, which code for tRNA, will also fold upon
formation and serve to bring amino acids to the ribosome later in the story. The mRNA
genes, on the other hand, code for messenger RNA, which serve as instructions that dictate
the amino acid sequence of a particular polypeptide. In this case, the mRNA gene is the
glucose transporter gene. Before leaving the nucleus, but after transcription, the mRNA
gets modified. A 5’ cap is added as well as a tail of 50 or more A nucleotides (poly-A tail) on
the 3’ end both by distinct enzymes. The ribosomal subunits, tRNAs and mRNAs will be
exported from the nucleus through the nuclear pore. The process of translation, which is
the reading of the mRNA and subsequent building of the polypeptide begins when the small
subunit of the ribosome randomly collides with the 5’ cap of the mRNA and specifically
binds to it. All collisions of molecules the size of proteins and smaller are random. However,
the binding is very specific and is based on shape and charge of the two molecules. The
small subunit will scan along the mRNA until it reaches the start codon. Codons can be
thought of as the words of the nucleic acid language or the words of the books (DNA). All
the codons are only three-letters and since there are only four letters (A,T,C,G) there are
only 64 possible words. The first codon or start codon is almost always AUG. Next, a tRNA,
called the initiator tRNA, containing a very specific complementary sequence of nucleotides
to the start codon (3’-UAC-5’) known as the anticodon will collide and bond via
complementary base-pairs to the start codon. This particular tRNA with the 3UAC5
anticodon always carries with it the amino acid methionine. Therefore, the first amino acid
of most proteins is methionine. The large subunit binds next to the small subunit and
mRNA placing the initiator tRNA into what is called the p-site. There is a second site over
the next codon called the a-site. The next tRNA will collide with the codon in the a-site and
it complementary will bind bringing the next very specific amino acid according the mRNA
instructions. The ribosome will then catalyze formation of the peptide bond between the
two amino acids. The carboxyl side of the methionine will be attached to the amino side of
the second amino acid. Therefore the polypeptide is being made from the N- toward the Cterminus. The ribosome will now translocate one codon. This will slide the a-site over to
the third codon and the p-site over the second codon. The initiator tRNA, bound to the start
codon, will now be out of the ribosome and will fall off the mRNA. The tRNA with the
dipeptide will be in the p-site, and the a-site will be open to receive the next tRNA. This will
continue and the nascent polypeptide will emerge from the ribosome. Since this is a
membrane protein, the first 20 or so amino acids will have a very specific sequence known
as the ER localization sequence or ERLS. There is a ribonucleoprotein (RNA-protein
complex) known as the signal recognition particle (SRP), which will collide with this
nascent polypeptide and bind to it. This entire complex containing the SRP, mRNA,
ribosome and tRNAs will collide with the translocon of the rough ER. The translocon has a
ribosome receptor and an SRP receptor. It also contains pore proteins that will allow the
growing polypeptide to cross the membrane and enter the ER. The SRP will hand off the
polypeptide to the pore proteins and the ribosome/tRNA will continue translation as the
polypeptide is thread through the translocon. As the polypeptide enters, it will be
simultaneously glycosylated by a number of ER resident enzymes called
glycosyltransferases. To be glycosylated means to have oligosaccharides added on at very
specific locations, which will turn the polypeptide into what is called a glycoprotein. The
oligos are part of the structure and therefore critical in terms of function. Glucose
transporter protein is a membrane protein and therefore has a very special amino acid
sequence within the polypeptide that will signal the translocon to slide this section of the
polypeptide into the membrane. This is where the protein becomes a membrane protein.
Eventually the ribosome will reach the stop codon for which there is no complementary
tRNA. Instead there is a protein called release factor that will bind to the stop codon and
cause the ribosome to dissociate from the mRNA. The membrane protein is now completed.
In order to get it out to the actual cell membrane, protein of the ER will pinch off a
membranous transport vesicle that will contain various membrane and soluble proteins for
delivery to the golgi. Kinesin, a motor protein found in the cytosol, will bind to specific
proteins in the membranes of the vesicles and walk the vesicles along mictotubules using
ATP as a source of energy as these structure are too large and move to slowly to simply
allow random motion for proper delivery. Once many vesicles arrive at the Golgi they will
fuse forming was is called the ‘cis’ or receiving cisterna. Within the Golgi, oligosaccharides
are modified by Golgi resident enzymes and proteins are sorted so that they go to their
proper final destination. New cis cisternae will form behind the old one moving the old one
forward in line. It is now a medial cisternae. The Golgi is in fact composed of many of these
membranous pancake-like stacks. Eventually the medials will continue being moved
forward until becoming a trans cisterna, which is last in line. This model where the cis
eventually matures into the trans is known as the cis maturation model. The trans cisterna
will break up into a number of different types of vesicles. The glucose transporter will be in
the membrane of a secretory vesicle destined for the cell membrane. Kinesin will again
walk it out there. Upon arrival a number of proteins will serve to fuse the vesicle with the
cell membrane. The secretory vesicle membrane will now become part of the cell
membrane, all the proteins inside the vesicle will be secreted outside the cell, and glucose
transporter will finally be where it needs to be and serve to transport glucose into the cell
(facilitated diffusion).