Protein Sorting Overview
Sections
KEY MECHANISMS OF PROTEIN TRANSPORT
- Gated transport: energy-dependent (nuclear pores)
- Translocation across membranes: mediated by protein translocators (mitochondrion, post-translational and cotranslational import into ER or peroxisomes)
- Vesicular transport: proteins do not cross membranes (from ER to Golgi, through Golgi stacks or to plasma membrane)
PROTEIN TRANSLATION
• Synthesis of almost all proteins begins in the cytoplasm
• 3 possible outcomes depending on signal sequence (or lack thereof):
i. remain in cytosol ii. post-translational import into organelle (mitochondrion, peroxisome, nucleus, ER) iii. cotranslational import to ER
SIGNAL SEQUENCES
• Short amino acid sequences at the terminal end of proteins (address labels)
• 15-60 residues long
• Direct proteins to specific organelles
• Proteins without signal sequences remain in the cytosol
MEMBRANE TOPOLOGY
• Compartments are topologically equivalent if molecules can get from one to another without having to cross a membrane
• ER, Golgi, vesicle, perinuclear space in nuclear envelope are all topologically equivalent
• Mitochondrion, nucleus and peroxisomes are topologically distinct
Full-Length Text
- In this tutorial we will learn how newly synthesized proteins in the cell are delivered to their sites of function.
Before we illustrate the major protein sorting pathways, let's draw a eukaryotic cell with its organelles.
- First, draw a plasma membrane.
- Label the cytosol and the extracellular space.
- Now, let's add the membrane enclosed organelles.
- First, draw a nucleus with a double-layered nuclear envelope.
- Next, erase a portion of the outer nuclear membrane.
- Now, draw the endoplasmic reticulum as continuous with the outer nuclear membrane.
- Draw the Golgi apparatus as a series of stacked cisternae.
- Now, draw a representative mitochondrion as follows:
- Draw an oval-shaped outer membrane, and an inner membrane with numerous folds called cristae.
- Finally, draw a peroxisome as a circle with a single membrane.
Now that we've drawn out a simplified eukaryotic cell, start a table to learn the three key mechanisms of protein transport.
- Denote that they include:
- Gated transport
- Translocation across membranes
- Vesicular transport
We will address where and when proteins use each of these transport mechanisms.
Now, return to our diagram.
- First, draw messenger RNA (mRNA) in the cytoplasm as a wavy line.
- Draw two ribosomes in the process of translating our mRNA transcript.
- As a review, DNA is transcribed in the nucleus to RNA, which is translated in the cytosol by ribosomes.
Now, let's address translation.
- Write that the synthesis of almost all proteins begins in the cytoplasm cytosol.
- As an exception, some proteins are synthesized within the mitochondria, but we will not address them in this tutorial.
- Now, draw a protein emerging from the ribosomes.
- Show that from here, our newly synthesized protein has three possible outcomes.
Let's illustrate them as follows:
- For outcomes 1 and 2: draw completely synthesized, folded cytosolic proteins. We will distinguish them shortly.
- For outcome 3: redraw the ribosomes and show that the mRNA transcript and the nascent protein remain bound to them.
- Now, draw signal sequences at the terminal end of the proteins in outcome two and three, which are short amino acid sequences – they are address labels that direct proteins to different organelles within the cell where they are recognized by specific signal receptors.
Let's learn some key features of signal sequences before we move on.
- Write the following regarding signal sequences:
- They are typically 15-60 residues long.
- They direct proteins to specific organelles within the cell.
- Proteins without signal sequences remain in the cytosol.
- The protein in outcome 1, which lacks a signal sequence, folds to its native conformation in the cytosol.
Now, return to our diagram to show the possible destinations for the proteins in outcomes 2 and 3.
- Show that the fully synthesized, folded protein in outcome 2 could travel to any of the following organelles:
- Nucleus, where it can pass through pores in its folded conformation.
- Mitochondrion, where it must unfold before crossing the membrane.
- Endoplasmic reticulum, where it enters unfolded.
- Peroxisome, where it crosses the membrane partially folded.
- It could also travel to chloroplasts in plant cells, but we will not discuss this here.
- How does this newly synthesized protein enter these membrane bound organelles?
- It uses the key mechanisms for transport that we listed at the beginning of this tutorial.
Let's illustrate them now.
- Indicate that the protein may cross the nuclear envelope using gated transport.
- Pores in the nuclear envelope form gates through which molecules can travel in both directions.
- Denote that gated transport in the nucleus is energy-dependent.
- Proteins that are imported into the nucleus include those involved in DNA transcription and replication.
- Indicate that a protein can use translocation to enter each of the following organelles:
- Mitochondria
- ER
- Peroxisome
- The protein refolds to its appropriate conformation in these compartments.
- Now, denote that translocation is mediated by protein translocators, which span the peroxisomal membrane, and both outer and inner membranes mitochondria.
Finally, return to our diagram so we can address outcome 3.
- Show that the signal sequence on the partially synthesized protein in outcome 3 directs the entire complex to the ER membrane where it docks.
- Show that this process also requires a protein translocator embedded in the ER membrane.
Now, let's differentiate the timing of translocation for the proteins in outcomes 2 and 3. We will use symbols for cotranslational and posttranslational transport for clarity.
- Indicate that in outcome 2, translocation is post-translational.
- Thus, proteins can enter the ER before or after translation is complete.
- Indicate that in outcome 3, translocation is cotranslational. In other words, ribosomes continue synthesizing the protein as it enters the translocator, unlike the translocation of outcome 2, which was post-translational.
This brings us to our final transport mechanism: vesicular transport.
- To illustrate this, draw a vesicle in each of the following locations:
- Between the ER and Golgi
- Adjacent to the cisternal stacks of the Golgi
- Between the Golgi and the plasma membrane.
- Now, draw an arrow from the ER to the first adjacent vesicle to show that proteins travel from the ER in membrane-bound vesicles.
- Use another arrow to show that this vesicle delivers proteins to the Golgi apparatus, where they are processed and modified.
- In general, transport vesicles carry cargo derived from the lumen of one compartment and fuse with their destination compartment to deliver it.
- Again, use arrows to show that the vesicle travels through the Golgi stacks in this manner.
- Draw a final arrow to show that the fully modified protein buds from the Golgi in a final vesicle.
- Denote that in vesicular transport, proteins do not have to cross membranes as they do in the other transport mechanisms.
- The lumens of organelles mix as transport vesicles fuse with them.
- Thus, proteins avoid the thermodynamic expense of crossing a membrane.
This brings us to our final point.
- Write that compartments are topologically equivalent if molecules can get from one to another without having to cross a membrane.
- To illustrate this, shade the lumen of the endoplasmic reticulum, the Golgi apparatus, and the vesicles that bud from and fuse with them a single color.
- These spaces are topologically equivalent because molecules can move between them without crossing a membrane.
- Shade the space between the outer and inner membrane of the nuclear envelope the same color.
- As we illustrated, this space is continuous with the ER lumen.
- Finally, shade the mitochondrion, nucleus and peroxisomes with three different colors to illustrate that they are topologically distinct.
- Molecules must cross membranes to enter these compartments.