University of Texas Medical Branch

Cell Biology Graduate Program

Formation of the Early Endosome.

After entering the cytoplasm, the endocytotic vesicle loses its clathrin coat.  Then it quickly fuses with other such vesicles in a process called "homotypic" (same type) fusion.  Each vesicle has a pair of SNARE proteins, (a v-SNARE and a t-SNARE) which are used in the initial process (these are called cis-SNAREs).  In homotypic fusion, the act is primed by the untangling of the SNARE pairs by N-ethylmaleimide-sensitive factor (NSF) which also uses a Soluble NSF attachment protein (SNAP).

Then, the untangled t-SNAREs are stabilized by another factor called LMA-1. The t-SNARE of one vesicle is aligned with the v-SNARE of another. In the meantime, specific docking/tethering factors bring the vesicles into closer alignment using their surface rab5-GDP. One of these is Rabaptin-5, which occurs with a nucleotide exchange factor (Rabex-5) that can activate rab5. It also has two  GTPase that could tether two endosomes together.   Another factor is Early Endosome Associated Protein (EEA-1) which are "coiled coils'.  Its C terminus has a FYVE finger domain that binds phosphatidylinositol-3-phosphate in endosome membranes.  EEA-1 not only tethers endosomes, it activates endosome fusion. This process is seen in the following cartoon

Thus, markers for early endosomes include:

• EEA-1 proteins
• rab5-GDP
• pH around 6.0

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Early endosome recycles receptors to the surface

Early endosomes have lowered pH (5.9-6) and this can release the receptor and ligand.  The receptor may be recycled to the surface by vesicles that bud from the endosome and then target the plasma membrane. After these recycling vesicles fuse with the plasma membrane, the receptor is returned to the cell surface for further binding and activity.  Then, the early endosome converts to a late endosome.

What happen to each receptor in the endosome?

The exact fate of the receptor in the membrane appears to vary with the cell. It can also be degraded. However, some receptors move to the Golgi complex to be added back to membranes in the Trans Golgi region. This would recycle the receptor. This process is similar to the process by which lysosomal enzyme receptors are recycled. In many cases, the receptor is sent back to the plasma membrane after a transport vesicle buds from the endosome. This event is shown in the above photograph. Willingham and Pastan used ferritin labeled antibodies to the extracellular domain of the receptor to follow recycling (transferrin receptor). This photograph was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985 As stated in the above paragraphs, in cases where the receptor is recycled from a budding endosome, the endosome itself is called an "early endosome". In other words, the pH has dropped just enough to allow the ligand to drop off, however, the receptor is not degraded. This keeps it intact so it can be recycled back to the membrane.

Formation of a late endosome

Late endosomes are formed as the pH continues to drop to 5-6.0.  Also, clathrin-coated vesicles from the Trans Golgi Network carry digestive enzymes to the late endosome and fuse with these structures, releasing their contents. The late endosome thus becomes a degradative body and also acquires the marker for mannose 6 phosphate receptor "MPR+

It changes its rab surface marker to rab7-GTP, probably to accommodate the new targeting vesicles with which it will fuse. This means that the late endosome can be identified by the presence of the rab7.

Late endosomes include multivesicular bodies and contain whorls or vesicles of membranes inside. They also contain an unusual lipid which has become another marker for this stage.  The lipid is called lysobisphosphatidic acid (LBPA) which has a larger head group than tail. Its structure enables it to be inserted into highly curved membranes, like the membrane whorls.  It is believed that this allows retention and binding of specific molecules in the whorls by lipid-protein; lipid-lipid interactions.  One type of molecule is cholesterol and it is believed that this is an important site for cholesterol accumulation.

For more information about the role of LBPA, see: Kobayashi, T, Beuchat, M.H, Lindsay, M, Frias S, Palmiter, R.D., Sakuraba, H, Parton, R.G, and Gruenberg, J.  Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biology 1: 113-118. 

Late endosomes function to degrade many proteins and lipids.  They also are responsible for returning the MPR receptors back to the Trans Golgi network.  They recycle these by budding off membranes that carry back the receptors and target the Trans Golgi membranes for fusion.  After fusion, the MRP receptors are available to capture and sort new degradative enzymes for future trafficking to the late endosomes.

To summarize, markers for late endosomes include:

• rab7-GDP
• LBPA
• MPR+

Late endosomes fuse with lysosomes.

Finally, late endosomes may not be able to digest all the material. Therefore, the next step is a fusion of late endosomes and lysosomes creating a hybrid organelle.  Residual heavily glycosylated lysosomal associated membrane proteins (LAMPs) may thus be transmitted to lysosomes.  LAMPs then become a marker for a late endosome or a lysosome. Since lysosomes do not have MPR receptors (they have all been sent to the Golgi), one could distinguish the lysosome and the late endosome on the basis of labeling for MPR.  Thus, fusion begins after the MPR have been sent back to the Trans Golgi.

The steps involved in forming late endosomes and lysosomes are drawn in this cartoon. Note that lysosomes continue to communicate with late endosomes and may deliver important material back to this group of organelles.  Lysosomes are considered the end product of endocytosis.  Thus,  lysosomes do not communicate directly with the Trans Golgi (and hence the plasma membrane).  However, they could communicate with upstream structures by way of the retrograde transport to the late endosome.

To summarize, markers for lysosomes include:

• LAMP+
• acid hydrolases
• MPR negative
• NPC1 (in normal cells)

For more information, see: Mukherjee , S, and Maxfield, F.R. Cholesterol: stuck in traffic.  Nature Cell Biology 1: E37-E38.

Receptor-mediated uptake of LDL receptors: a model for studies of trafficking and defects.

Cholesterol bound to Low density lipoproteins (LDL) is taken up by cells so that cholesterol can be used in construction of membranes, etc.  In this case the receptor is recycled and the ligand (LDL-cholesterol) is metabolized so the free cholesterol can be released and used by the cell. There are two genetic mutations that cause either no uptake of LDL receptors or uptake and accumulation of cholesterol in late endosomes.  We will look at these diseases to learn more the importance  of Key elements in receptor mediated endocytosis are.

First, this cartoon summarizes the entire process of endocytosis of LDL-cholesterol bound to LDL receptors. (taken from Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994 ). To review, after the ligand and receptors are collected in the coated pits, which then form coated vesicles, the clathrin coat is removed and the vesicle fuses with forming endosomes. The early endosome allows recycling of the LDL receptor.  The late endosome/lysosome is the site of accumulation of cholesterol followed by hydrolysis.  Free cholesterol is then available to be used by the cell.   

How do receptor mediated endocytosis and LDL receptors help reduce our serum levels of cholesterol?

Cholesterol Stuck in Traffic: What we can learn from studies of Nieman Pick type C disorder.

Recent studies point to an important protein involved in cholesterol efflux from the late endosomes, called NPC1.  This protein has a putative steroid binding domain. It may be involved in transport of cholesterol to the Trans Golgi Network and then to plasma membranes.  Hence it may be important in the addition of cholesterol to membranes where it is needed.  It may also be a sensor for sites of accumulated cholesterol.

It got its name from a disease, which is caused by an autosomal recessive mutated gene in which this NPC1 protein is not normal.  This is called Niemann Pick type C disorder. The mutation results in an accumulation of cholesterol in late endosomes which also expand, filling with whorls of membranes.   

Where is NPC1 protein normally found?  Studies by Neufeld et al (J Biol Chem. 274: 9627-9635 (1999)  have shown however that NPC1 protein in normal fibroblasts is in the lysosomes (identified by their LAMP marker and absence of MPR).  Yet, this same compartment did not contain large amounts of LDL-cholesterol.  Thus, Neufeld et al  suggested that lysosomes send the NPC1 back to late endosomes by retrograde traffic (see above cartoon).  Perhaps this is stimulated by the concentration of cholesterol in the late endosomes.  As Mukherjee and Maxfield ( Cholesterol, Stuck in Traffic, Nature Cell Biology 1: E37-E38) point out, this route would then give direct access to the Trans Golgi network to allow trafficking of cholesterol to sites where the cholesterol is needed.  Lysosomes don't have routes to these sites, so they may have to communicate to the rest of the cell via late endosomes.

Where is cholesterol needed?  Wherever there is a need for more structural stabilization of membranes, for example wherever there are coated pits, particularly clathrin coated pits.

Kobayashi et al [Kobayashi, T, Beuchat M-H, Lindsay, M, Frias S, Richard D. Palmiter, H Sakuraba, R.G. Parton, and J. Gruenberg  Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nature Cell Biology 1: 113-116 (1999).] studied fibroblasts from Nieman Pick Type C disorder (NPC) and found that not only was cholesterol stuck in the late endosome, mannose 6 phosphate receptors (MPR) are also stuck in this part of the trafficking pathway.  Immunolabeling for MPR and CD63 (a marker for late endosomes) showed normal distribution in late endosomes and Golgi regions in normal cells as well as in cells from another disease state, Tay Sach's.  However,  cells from patients with NPC had cholesterol and MPR almost exclusively in late endosomes.  

These studies continued by using a drug (U18666A) to mimic the disease state and they found that they could cause both an accumulation of the cholesterol in late endosomes, and also a block in the transport of MPR back to the Golgi complex.  Hence, MPR was detected exclusively with structures labeled for late endosome markers including LBPA and rab7.

What is the significance of these studies?  The NPC1 protein may function as a cholesterol sensor/binding protein. An accumulation of cholesterol in the late endosomal compartment may stimulate retrograde transport from lysosomes to endosomes bringing the NPC1 protein to the endosomes.  There, the cholesterol can be sorted and further distributed to membranes throughout the cell, as needed.  If the NPC1 protein is not functional, then cholesterol accumulates in the membrane whorls seen the late endosomes.  This appears to block retrograde transport from the endosome to the Golgi complex, as evidence by the accumulation of the MPR.  Hence, not only is cholesterol "stuck in traffic", so is the mannose 6 phosphate receptor (MPR).  It appears to be stuck as a consequence of the cholesterol accumulation.   There is too little known about the return trip to the Golgi complex. However, stay tuned for future information about the role of microtubules and associated motor proteins.  

In the meantime,  Mukherjee and Maxfield (Nature, Cell Biology, 1: E37-E38 (1999)  speculate that perhaps the accumulation of cholesterol in the late endosome makes the membranes less elastic and thus, the return vesicles to the Trans Golgi Network cannot bud and form.  There may be broader effects on trafficking as well.  Return to the page on membrane architecture for more information about the role of cholesterol in membrane structure and function.

Below is a modified cartoon showing the site of the block in trafficking in Nieman Pick C disorder.

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Supposing a cell is stimulated with two hormones (and it has receptors for both), or a hormone and a growth factor. Do both ligands enter via the same packages, or is there a selection for one particular type of ligand in a coated pit. This is a perfect question for cytochemistry with different types of labels attached to different ligands. For example, one can use colloidal gold, ferritin, peroxidase and detect as many as 2-4 ligands on a given cell. 

This figure shows that multiple ligands can enter the cell in the same coated pit. Furthermore, the vesicles will carry them to the same receptosomes. The photo shows co-detection of ligands as diverse as Epidermal growth factor, vesicular stomatitis virus, or alpha 2 macroglobulin. Labeling molecules (signalling molecules) included gold, peroxidase, ferritin, or the virus itself. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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This cartoon shows the sorting of proteins destined for secretion into vacuoles in the trans Golgi Complex. Note that lysosomal enzymes are sorted into another compartment, thanks to the mannose 6 phosphate receptor. For review, see the Return to Menu

What is Regulated exocytosis ?

The above cartoon also shows the process of exocytosis. We defined regulated secretion initially when we talked about the Golgi complex . Regulated secretion requires a stimulus, usually from the outside. The process may begin by the binding of a ligand to a specific receptor. This activates the receptor which turn activates a cascade of events (second and third messengers) leading to the release of the product. If the product is stored in a secretory granule, it is released by a process called "exocytosis". Therefore, the process of receptor mediated endocytosis and exocytosis can and does occur simultaneously. One should be able to recognize the cytological signs of both processes in the same cell. Click this link to see an electron micrograph of exocytosis.

Distinguish profiles showing exocytosis and endocytosis

As we said earlier in this presentation, clathrin coated pits serve like a flat "basket", stabilizing the area to be internalized. They are not exclusive to the plasma membrane. However, it is important to be able to distinguish them from exocytosis profiles associated with the plasma membrane.
For example, this electron micrograph is showing the process of exocytosis . The process begins by fusion of the membranes at the peripheral pole of the granule. Then an opening is created which widens to look like an omicron figure. This opening allows the granular material to be released. The membrane is now part of the plasma membrane and any proteins carried with it can be incorporated into the plasma membrane. Note that there is no coating on the membrane. This figure was taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing Third Edition, 1994