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PROTEIN SORTING IN THE
ENDOSOMAL-LYSOSOMAL SYSTEM
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Juan S. Bonifacino, PhD, Head,
Section on Intracellular Protein Trafficking Rafael
Mattera, PhD, Staff Scientist Cecilia
Arighi, PhD, Postdoctoral Fellow Cecilia Bonangelino, PhD, Postdoctoral Fellow Katy
Janvier, PhD, Postdoctoral Fellow Satoshi
Kametaka, PhD, Postdoctoral Fellow Bong-Yoon
Kim, PhD, Postdoctoral
Fellow Stephane
Lefrançois, PhD, Postdoctoral Fellow Wolf
Lindwasser, PhD, Postdoctoral Fellow José
Martina, PhD, Postdoctoral Fellow Peter
McCormick, PhD, Postdoctoral Fellow Rosa Puertollano,
PhD, Postdoctoral
Fellow William
Smith, PhD, Postdoctoral Fellow Hadiya Watson, PhD, Postdoctoral Fellow Joost
Drenth, MD, Guest Researcher Xiaolin Zhu, RN, Technician Lisa Hartnell, BS, Research Assistant |
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We investigate the molecular mechanisms that
control the sorting of transmembrane proteins in the endosomal-lysosomal
system. Sorting events such as rapid internalization of receptors from the
plasma membrane, targeting to lysosomes and lysosome-related organelles, and
delivery to the basolateral plasma membrane domain of polarized epithelial
cells are all mediated by recognition of specific signals in the cytosolic
domains of the transmembrane proteins by adaptor proteins associated with the
cytosolic face of membranes. Among these proteins are the heterotetrameric
adaptor protein (AP) complexes AP-1, AP-2, AP-3, and AP-4 and the monomeric
GGA1, GGA2, and GGA3 proteins (see Figure 2.1). Mutations in AP-3 are the
cause of the pigmentation and bleeding disorder Hermansky-Pudlak syndrome
(HPS) type 2. Our current work focuses on elucidating the structure,
regulation, and physiological roles of the AP complexes and GGA proteins as
well as on investigating the possibility that defects in or interference with
these proteins underlie certain human diseases. Role
of the GGA proteins in sorting to lysosomes Mattera, Puertollano, Smith, Bonifacino;
in collaboration with Hurley, McPherson, Miller, Ritter, Sidhu Over the past year, we have continued our
studies on the structure and function of the GGAs.
These proteins are composed of four domains
named VHS, GAT, hinge, and GAE. The VHS domain is a recognition module for a subset
of “dileucine-based” sorting signals present in the cytosolic domains of the
mannose 6-phosphate receptors (MPRs) that sort acidic hydrolases to
lysosomes. The GAT domain binds to GTP-Arf and targets the GGAs to the trans-Golgi network (TGN). The hinge
domain recruits clathrin, and the GAE domain binds to accessory factors
involved in vesicle budding and fusion and in interactions with the
cytoskeleton. These properties indicate that the GGAs function as
Arf-dependent adaptors for the recruitment of clathrin to the TGN and for
sorting MPRs and their cargo, the lysosomal hydrolases, to lysosomes. Over
the past year, we identified a canonical peptide motif that mediates
interaction of a cohort of accessory proteins with the GAE domain of the GGAs
and the related “ear” domains of the gamma subunit of AP-1. The definition of
this motif has allowed us to identify novel accessory proteins involved in
protein sorting at the TGN. In addition, we have identified novel binding
partners for the GAT domain, including ubiquitin, the tumor susceptibility
gene 101 product (TSG101), and Rabaptin-5; binding of these proteins occurs
through distinct but overlapping sites on the GAT domain. The identification
of these binding partners indicates that the GGAs engage in a wide network of
interactions with components of the protein trafficking machinery. Conjugation of ubiquitin to proteins generally
functions as a signal for targeting to degradative organelles such as
proteasomes and lysosomes. TSG101 is a component of the endosomal machinery
that mediates ubiquitin-dependent transport to lysosomes. The fact that the GGAs
interact with ubiquitin and TSG101 suggests that the GGAs may have an
additional role as ubiquitin adaptors. Indeed, we found that depleting GGA3
and GGA1 with small interfering RNAs (siRNAs) resulted in accumulation of
internalized epidermal growth factor (EGF) receptors in enlarged early
endosomes and partially blocked their delivery to lysosomes. EGF receptors
are normally downregulated by internalization and targeting to lysosomes
through a process that involves conjugation of ubiquitin to their cytosolic
tails. The role of the GGAs in allowing movement of EGF receptors to
lysosomes depended on their ability to bind to ubiquitin, thus demonstrating
that the GGAs also function as ubiquitin adaptors. Experiments are under way to determine whether defects in the
GGAs underlie any lysosomal storage disorders. Bonifacino JS. The GGA proteins: adaptors on
the move. Nat Rev Mol Cell Biol
2004;5:23-32. Mattera R,
Puertollano R, Smith WJ, Bonifacino JS. The tri-helical bundle subdomain
of the GGA proteins interacts with multiple partners through overlapping but
distinct sites. J Biol Chem
2004;279:31409-31418. Mattera R, Ritter B, Sidhu SS, McPherson PS,
Bonifacino JS. Definition of the consensus motif recognized by gamma-adaptin
ear domains. J Biol Chem
2004;279:8018-8028. Miller GJ, Mattera R, Bonifacino JS, Hurley
JH. Recognition of accessory protein motifs by the gamma-adaptin ear domain
of GGA3. Nat Struct Biol
2003;10:599-606. Role
of AP complexes and retromer in protein trafficking Janvier, Kato, Boehm, Martina, Kim,
Arighi, Hartnell, Lefrançois; in collaboration with Haft, Ooi, Venkatesan In addition to the GGAs, the heterotetrameric
AP complexes AP-1, AP-2, AP-3, and AP-4 play important roles in protein
trafficking. They recognize both tyrosine-based signals as well as a subset
of dileucine-based sorting signals distinct from those recognized by the GGAs.
The identity of the AP subunits that harbor the binding sites for the
dileucine-based sorting signals has, however, remained elusive. We used a
yeast three-hybrid assay to demonstrate that the dileucine-based sorting
signals from the human immunodeficiency virus Nef gene product and the lysosomal membrane protein LIMP II
interact in a bipartite manner with combinations of the gamma and sigma-1
subunits of AP-1 and the delta and sigma-3 subunits of AP-3. These
observations thus revealed a novel mode of recognition of sorting signals by
AP complexes requiring the co-expression of two subunits from each AP
complex. This knowledge could be helpful for the design of agents that
interfere with the action of Nef as well as for explaining the sorting of
many important cellular proteins such as the Niemann-Pick type C protein and
the glucose transporter GLUT4, which also have dileucine-based sorting
signals. We have examined the physiological roles of AP
complexes in the sorting of proteins to lysosomes by using an RNA
interference approach. Although all four AP complexes are capable of binding
to subsets of tyrosine-based and dileucine-based sorting signals found on the
cytosolic domains of lysosomal transmembrane proteins, AP-2 is the most
important for sorting these proteins in cells. Given that AP-2 is associated
with the plasma membrane, the trafficking of lysosomal transmembrane proteins
must involve passage via the plasma membrane en route to lysosomes. AP-3 is
the next most important complex for lysosomal targeting. We recently
uncovered important aspects of the mechanism by which AP-3 is recruited to
membranes. Binding of AP-3 to membranes is regulated by the small GTP-binding
protein Arf. Release of AP-3 from membranes requires hydrolysis of GTP to GDP
on Arf. We have now found that a protein named AGAP1 binds to AP-3 and
activates GTP hydrolysis on Arf, thus effecting the dissociation of AP-3 from
membranes. We also found that the interaction of AP-3 with GTP-Arf is regulated
by an intramolecular interaction between the “ear” domain of the delta
subunit and the sigma-3 subunit of AP-3. These observations thus provide a
detailed understanding of the molecular mechanisms that control the function
of an AP complex. We also discovered that another protein
complex, referred to as the “retromer,” retrieves the MPRs to the TGN after
they release the lysosomal hydrolases in endosomes. Depletion of this complex
by RNA interference results in missorting of MPRs to lysosomes, secretion of
lysosomal hydrolases, and accumulation of undegraded materials in lysosomes, giving
rise to a lysosomal storage disorder similar to that observed in “I-cell”
disease. Arighi CN, Hartnell LM, Aguilar RC, Haft CR,
Bonifacino JS. Role of the mammalian retromer in sorting of the
cation-independent mannose 6-phosphate receptor. J Cell Biol 2004;165:123-133. Bonifacino JS, Glick BS. The mechanisms of
vesicle budding and fusion. Cell
2004;116:153-166. Janvier K, Kato Y, Boehm M, Rose JR, Martina
JA, Kim BY, Venkatesan S, Bonifacino JS. Recognition of dileucine-based
sorting signals from HIV-1 Nef and LIMP II by the AP-1 gamma-sigma1 and AP-3
delta-sigma3 hemicomplexes. J Cell Biol
2003;163:1281-1290. Lefrançois S,
Janvier K, Boehm M, Ooi CE, Bonifacino JS. An ear-core interaction
regulates the recruitment of the AP-3 complex to membranes. Dev Cell 2004;7:619-625. Nie Z, Bohm M, Boja ES, Vass WC, Bonifacino
JS, Fales HM, Randazzo PA. Specific regulation of the adaptor protein complex
AP-3 by the Arf GAP AGAP1. Dev Cell
2003;5:513-521. Biogenesis
of lysosome-related organelles and the Hermansky-Pudlak syndrome Martina, Moriyama; in collaboration with
Ciciotte, Gwynn, Peters The characterization of the molecular
machinery involved in protein sorting is important for understanding the
pathogenesis of various metabolic and developmental disorders. An example is
the Hermansky-Pudlak syndrome (HPS), a genetically heterogeneous disease that
affects lysosome-related organelles such as melanosomes and platelet dense
bodies. We previously discovered that mutations in the gene encoding the
beta3A subunit of AP-3 are the cause of HPS type 2. Strikingly, mutations in
at least five other genes in humans and 14 genes in mice cause a similar
disorder. Most of the HPS genes identified to date by positional cloning encode
proteins of unknown function and with no recognizable homology to other
proteins. To gain insight into the nature of this sorting machinery, we have
undertaken a biochemical characterization of the novel HPS gene products. We
previously found that the protein products of the pallid, muted, and cappuccino genes are the components of
a complex named BLOC-1. This past year, we identified another component of
this complex, which is encoded by the reduced
pigmentation gene. In addition, we showed that the products of the pale ear and light ear genes are part of another complex, which we named
BLOC-3. The properties of BLOC-1 and BLOC-3 are consistent with their being
components of the molecular machinery for the biogenesis of lysosome-related
organelles. Ongoing studies on these complexes are likely to provide
additional insights into the pathogenesis of HPS. Ciciotte SL, Gwynn B, Moriyama K, Huizing M,
Gahl WA, Bonifacino JS, Peters LL. Cappuccino, a mouse model of
Hermansky-Pudlak syndrome, encodes a novel protein that is part of the
pallidin-muted complex (BLOC-1). Blood 2003;101:4402-4407.
Gwynn B, Martina JA, Bonifacino JS,
Sviderskaya EV, Lamoreux ML, Bennett DC, Kengo K, Huizing M, Helip-Wooley A, Gahl
WA, Webb LS, Lambert AJ, Peters LL. Reduced pigmentation (rp), a mouse model
of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1
complex. Blood 2004;104:3181-3189. Martina JA, Moriyama K, Bonifacino JS. BLOC-3,
a protein complex containing the Hermansky-Pudlak syndrome gene products HPS1
and HPS4. J Biol Chem
2003;278:29376-29384. Pathogenesis
of polycystic liver disease Drenth, Martina; in collaboration with
Jansen Polycystic liver disease (PCLD) is a
dominantly inherited condition characterized by the presence of multiple
liver cysts of biliary epithelial origin. In previous work, we identified a
defective gene in four large Dutch pedigrees; the gene encodes a protein that
we named hepatocystin. This past year, we demonstrated that hepatocystin
functions as the noncatalytic beta subunit of the endoplasmic reticulum
enzyme glycosidase II. The enzyme trims glucose residues from N-glycan chains
on newly synthesized glycoproteins, a reaction that is required for further carbohydrate
processing, polypeptide folding, and quality control. C-terminal–truncating
mutations of hepatocystin found in PCLD patients prevent assembly of
hepatocystin with the catalytic alpha subunit of glucosidase II and lead to
secretion of the mutant hepatocystin into the extracellular medium, resulting
in reduced levels of glucosidase II in cells from PCLD patients and virtually
undetectable glucosidase II in liver cysts. Our studies suggest that the
proliferation of the biliary epithelium observed in this disease is caused by
abnormal biogenesis in the endoplasmic reticulum of a regulator of biliary
epithelial cell proliferation or differentiation. Drenth JPH, Martina JA, Te Morsche RHM, Jansen
JBMJ, Bonifacino JS. Molecular characterization of hepatocystin, the protein
that is defective in autosomal dominant polycystic liver disease. Gastroenterology 2004;126:1819-1827. Drenth JPH, te Morsche RHM, Smink R,
Bonifacino JS, Jansen JBMJ. Germline mutations in PRKCSH are associated with
autosomal dominant polycystic liver disease. Nat Genet 2003;33:345-347. COLLABORATORS Steve Ciciotte, BS, The Jackson Laboratory, Carol Haft, PhD, Division of Diabetes, Endocrinology, and
Metabolism, NIDDK, James H. Hurley, PhD, Laboratory of Molecular Biology, NIDDK, Jan B.M.J. Jansen, MD, Peter S. McPherson, PhD, Gregory J. Miller, PhD, Laboratory of Molecular Biology, NIDDK, Zhongzhen Nie, PhD, Laboratory of Cellular Oncology, NCI, Chean Eng Ooi, PhD, Curagen, Luanne Peters, PhD, The Jackson Laboratory, Paul Randazzo, MD, Laboratory of Cellular Oncology, NCI, Carol Renfrew-Haft, PhD, Division of Diabetes, Endocrinology and
Metabolism, NIDDK, Brigitte Ritter, PhD, Sachdev S. Sidhu, PhD, Genentech, Sundararajan Venkatesan, PhD,
Laboratory of Molecular Microbiology,
NIAID, For further information, contact bonifacinoj@mail.nih.gov |
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