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56 Part I Molecular and Cellular Basis of Hematology

into large detergent-insoluble oligomers as a way of minimizing lipid- disease. Hurler syndrome is caused by a mutation in a hydrolase respon-
protein contact. This would prevent the entry of proteins into the sible for breakdown of glycosaminoglycans that prevents the hydrolase
vesicles and thus their traffic to more distal cisternae. There is evi- from acquiring the mannose-6-phosphate (M6P) modification, conse-
dence in support of both models. quently preventing targeting to lysosomes. Similarly, in I-cell diseases
undigested material accumulates in lysosomes because a mutation in
the enzymes that create the M6P modification, causes missorting of
Protein Trafficking to and Through the Golgi Apparatus lysosomal hydrolases.

Cargo proteins exit the ER in COPII-coated vesicles that enter the
ERGIC and are ultimately delivered to the cis-Golgi either in vesicles Autophagy: A Lysosomal Degradation Pathway
or along extended tubules. However, the mechanism whereby cargo
proteins move across the Golgi complex from cis to trans remains Autophagy, the most common name for macroautophagy, consists in
controversial. Two models have been proposed. The vesicular trans- the capture and degradation of cellular components and organelles.
port model contends that anterograde transport occurs in vesicles or Cellular material is sequestered inside double-membrane vesicles,
tubules that traffic cargo in an anterograde direction. The second called autophagosomes, and degraded upon fusion with lysosomal
suggests that there is a cisternal progression and maturation. This compartments. Raw precursors are then recycled for new biosynthe-
alternative model proposes that Golgi cisternae are not fixed structures ses. Constitutive autophagy serves to demolish damaged organelles
but move forward from the cis side to the trans side generating an or cytosolic components and contributes to the maintenance of cell
anterograde movement. As cisternae mature, resident Golgi proteins homeostasis.
that belong to more cis-like cisternae must be selectively pinched off Autophagy is also stress responsive. It accelerates the catabolism
in vesicles and trafficked back to the cis side of the Golgi stack. This of cellular components to sustain the demand of energy in adverse
would occur by COPI-mediated retrograde vesicular transport (see conditions and promotes cell survival. From yeast to human cells,
Fig. 5.4). Although which of these models is correct is currently starvation typically activates autophagy. Yeast has been a useful model
unclear, a majority of the experimental data supports the cisternal microorganism to identify the first autophagy genes (ATG) that
maturation model. In particular, technical progress in live-cell allowed the subsequent isolation of the mammalian counterparts. Atg
imaging provided evidence supporting a very dynamic nature of this proteins are involved in the basic mechanism of autophagy on which
organelle as expected by the progression/maturation model. a complex regulation has been superimposed in mammals to respond
to a wider variety of hormonal, environmental and intracellular
signals. An increasing body of evidence suggests that autophagy plays
SORTING EVENTS AT THE TRANS-GOLGI NETWORK an important role in development and cell differentiation by facilitat-
ing cell and tissue remodeling. Remarkably, the basis for erythrocyte
Overview maturation into reticulocytes, which involves mitochondria loss,
remained mysterious for decades, but is now known to be partly
The TGN is an important site of intracellular sorting, where proteins dependent on autophagy (mitophagy).
bound for lysosomes or regulated secretory vesicles are separated from Defects in constitutive autophagy compromise fitness of an organ-
those entering the constitutive pathway leading to the plasma mem- ism. As a consequence, defective autophagy increases susceptibility to
brane (see Fig. 5.4, pathways 6, 7 and 8). The secretion process is tumorigenesis, neurodegenerative disorders, liver disease, aging, inflam-
called exocytosis. The molecular basis for diversion of proteins into matory diseases and defective host defense against pathogens. However,
lysosomes and regulated secretory granules are described later. recent evidence suggests autophagy provides a survival advantage for
tumor cells in a hostile microenvironment. Thus, autophagy is regarded
as a target for tumor prevention and cancer therapy.
Sorting Into Lysosomes
Lysosomes are acidic (pH of approximately 5.0–5.5), membrane- Sorting Into Regulated Secretory Granules
bound organelles containing numerous hydrolytic enzymes designed
to degrade proteins, carbohydrates, and lipids. Soluble hydrolases are In regulated secretion proteins are condensed into stored secretory
selectively marked for sorting into lysosomes by phosphorylation of granules that require an appropriate stimulus for release from the cell
their N-linked oligosaccharides that creates the mannose-6-phosphate (see Fig. 5.4, pathway 7). After budding from TGN, the granule
sorting signal (M6P). On arrival at the TGN, the M6P-modified proteins are concentrated (up to 200-fold in some cases) by selective
hydrolase is bound by a cargo receptor, the M6P-receptor (M6P-R), removal of extraneous contents from clathrin-coated vesicles. Mature
which delivers it first to a “late endosomal compartment,” where the secretory granules are thought to be stored in association with micro-
low pH releases the hydrolase from the M6P-R. Subsequently, the tubules until the stimulation of a surface receptor triggers their
hydrolase is delivered to the lysosome, and the M6P-R is recycled exocytosis. One example of stimulus-induced exocytosis is the
from the endosomes through retromer-coated vesicles to the TGN to binding of a ligand to the T-cell antigen receptor (TCR) complex on
be reused (for simplicity the endosome to Golgi transport is not a cytotoxic T lymphocyte. Conjugation of a cytotoxic T cell with its
represented in Fig. 5.4). target causes its microtubules and associated secretory granules to
The motif responsible for targeting M6P-R to lysosomes is YSKV reorient toward the target cell. Subsequently, the granules are deliv-
and is recognized by all three distinct adaptor protein (AP) complexes ered along microtubules until they fuse with the plasma membrane,
(AP-l, -2, and -3) that contribute to delivery of cargo to lysosomes by releasing their contents for lysis of the target cell. Following release
linking cargo acquisition to vesiculation. Cargo recruitment occurs in of the granule contents, the granule membrane components are
a manner similar to that described for the COPI- and COPII-dependent internalized and transported back to the TGN, where the granule can
vesicles, except that the cytosolic coat complex is clathrin. In addition be refilled with cargo proteins.
to luminal hydrolases, lysosomes also contain a wide array of membrane
proteins that are targeted to lysosomes via one of two consensus motifs:
(1) YXXe, where X is any amino acid and e is any amino acid with a ENDOCYTIC TRAFFIC
bulky hydrophobic side chain and (2) a leucine-based motif (LL or LI).
Trafficking of these membrane-bound proteins to lysosomes is indirect, Overview
proceeding first to late endosomes or the plasma membrane before their
retrieval to lysosomes. Failure to accurately target lysosomal hydrolases Substances are imported from the cell exterior by a process termed
underlies two well-known human diseases, Hurler syndrome and I-cell endocytosis (see Fig. 5.4, pathway 9). Endocytosis also serves to recover