Targeting of the Lysosomal Tether, The HOPS Complex, Pubblico
Zlatic, Stephanic (2011)
Abstract
Localization of cytosolic protein between the cytoplasm and
lysosomal
membranes is critical for the biogenesis and maintenance of
lysosome molecular
composition and function. My dissertation examines how the
cytosolic tether,
HOPS complex, required for organelle fusion with the lysosome is
localizes to
subcellular domains in polarized and non-polarized mammalian cells.
Current
models of membrane traffic segregate vesicle formation machinery
from fusion
machinery spatially and functionally. In this model, tethers such
as the HOPS
complex localize to their target organelle through selective
recruitment from the
cytoplasm to the limiting membrane of the lysosome. However, this
model does
not account for the localization of HOPS complex subunits to early
and late
endosomes in mammalian cells described in my dissertation.
In my dissertation I hypothesize cytosolic HOPS complex
subcellular
localization is regulated by a vesicular coat-dependent mechanism
of membrane
traffic along the endocytic route. Here, I present biochemical
and
immunomicroscopy evidence that HOPS subunits interact and
colocalize with the
coat proteins AP-3 and clathrin and localize to sites reminiscent
of AP-3/clathrin
vesicle and clathrin plaque formation at early endosomes.
Biochemical
fractionation reveals HOPS complex subunits cofractionate with
clathrin-coated
vesicles. Furthermore, distribution of HOPS complex subunits to
both early and
late endosomes/lysosome subcellular locations is rapidly perturbed
by acute
inhibition of clathrin function through a chemical-genetic strategy
in non-
polarized cells. In polarized cells of neuronal origin, HOPS
complex subunits
localize to the tips of neurites. HOPS subunit localization to the
proximal region
of neurites decreased following acute chemical-genetic disruption
of clathrin
function.
Together, these data lead me to propose a novel model of HOPS
localization in mammalian cells in which HOPS complexes localize to
sites of AP-
3/clathrin vesicle formation and flat clathrin plaques at early
endosomes for their
delivery as cargo to the late endosome/lysosome either through
vesicular
transport and/or endosomal maturation. My findings provide novel
insight into
the possible roles that HOPS complex subunits may have in
regulation of
lysosome and lysosome-related organelle trafficking in mammalian
cells. Overall,
my dissertation challenges the current conception that vesicle
formation coats
and vesicle fusion tethers are spatially and functionally
segregated.
Table of Contents
Table of Contents
Page Number
Chapter I. General
Introduction.....................................................1-56
Overview...................................................................................2
Significance............................................................................2-5
Section 1. The Importance of Protein Targeting to the
Lysosome is Critical for the Maintenance of
Cellular
Function....................................................5-9
Section 2. Fundamentals of Molecular Complexes
Required for Lysosome Protein Localization........9-15
Section 2.01 ARFs and
Adaptors.................................9-11
Section 2.02 Clathrin: Plaques, and
Vesicles.............11-13
Section 2.03
SNAREs................................................13-14
Section 2.04 Rabs and
Tethers..................................14-15
Section 3. Comparative Molecular and Cell Biology
of the HOPS
Complex..........................................15-20
Section 4. Models of Membrane Trafficking Pathways
Along the Endocytic
Route..................................20-24
Section 4.01 Vesicular Model of Membrane
Traffic to the Lysosome.........................21-23
Section 4.02 Maturation Model of Lysosome
Traffic
..................................................23-24
Section 5. The Importance of the HOPS Complex for
Lysosome and Lysosome-Related Organelle
Function.............................................................24-30
Section 6. Comparative Phylogeny of S. cerevisiae
and
Mammalian HOPS-Dependent Mechanism
and the Central Contribution of This
Dissertation........................................................30-34
Section 7.
Figures...............................................................35-56
Figure 1. Organelles Maintain Unique Protein
Combinations Through Protein
Localization and Membrane Transport
Mechanisms...............................................35-36
Figure 2. Two Models of HOPS Complex Localization
to a Late Endosome/Lysosome
Compartment.............................................37-38
Figure 3. Model of Vesicle
Transport.........................39-40
Figure 4. GTP-ARF Recruits Cytosolic Adaptor to
Membranes......................................................41
Figure 5. Adaptor Protein
Complexes.............................42
Figure 6. Clathrin
Structures.....................................43-44
Figure 7. SNARE
Interactions....................................45-46
Figure 8. Rab
GTPase......................................................47
Figure 9. HOPS and CORVET
Organization....................48
Figure 10. Model of HOPS/AP-3 Interaction
in the yeast S.
cerevisiae................................49
Figure 11. Homologous Structures in HOPS Subunits
and Clathrin Heavy
Chain..............................50
Figure 12. Vesicular and Maturation Pathway
of Lysosome
Delivery.................................51-52
Figure 13. Rab
Conversion.........................................53-54
Figure 14. Notch Activation and Degradation
Pathways...................................................55-56
Chapter II. Clathrin-Dependent Mechanisms Modulate the
Subcellular Distribution of Class C Vps/HOPS Tether
Subunits in Polarized and Nonpolarized
Cells............................57-125
Abstract...................................................................................58
Introduction.......................................................................59-62
Results................................................................................62-76
Subunits of the Vps class C/HOPS tethering
complexes associate with clathrin-AP-3 adaptor
subunits....................................................................62-68
Vps class C and HOPS subunits are present in
clathrin-coated
organelles.........................................68-72
Acute perturbation of clathrin alters Vps
class C/HOPS subunit subcellular
distribution..........72-75
Clathrin and Vps class C proteins are targeted
to neuronal processes by clathrin-dependent
mechanisms..............................................................75-76
Discussion..........................................................................77-84
Materials and
Methods......................................................84-93
Acknowledgements..................................................................93
Figures..............................................................................94-125
Figure 1. Association of Class C Vps/HOPS
Subunits with the Adaptor Complex
AP-3............................................................94-96
Figure 2. Clathrin Heavy and Light Chains
Associate with Class C Vps/HOPS
Subunits...................................................97-100
Figure 3. Vps Class C/HOPS are Present in
AP-3, Clathrin, Rab5 and Rab7b-
Compartments.........................................101-102
Figure 4. Vps Class C/HOPS Subunits Localize
to Discrete Domains of Enlarged Early
Endosomes..............................................103-104
Figure 5. Vps Class C/HOPS Subunits Cosediment
with Clathrin-Coated Vesicles.................105-106
Figure 6. In Vivo Chemical-Genetic Disruption of
Clathrin Chains Rapidly Redistributes
Vps18......................................................107-108
Figure 7. Chemical-Genetic Disruption of Clathrin
Chains Affects Vps Class C/HOPS Subunits
and Coat
Distribution..............................109-110
Figure 8. Chemical-Genetic Disruption of Clathrin
Affects Vps33b Distribution in Rab5
and Rab7b Endosomal Compartments......111-112
Figure 9. Polarized Distribution of Clathrin and
Vps Class C Proteins in Neuronal Cells.....113-114
Supplementary Figure 1. The Vps33b-Interacting
Potein Spe39 does not Associate with
Clathrin or AP-3 Positive Organelles.........115-116
Supplementary Figure 2. Vps Class C/HOPS
Subunits Cosediment with Clathrin-
Coated
Vesicles.........................................117-118
Supplementary Figure 3. Chemical-Genetic
Disruption of Clathrin Chains Affects
Coat
Distribution.....................................119-120
Supplementary Figure 4. Chemical-Genetic
Disruption of Clathrin Affects Vps33b
Distribution in Endosomal
Compartments.........................................121-122
Supplementary Figure 5. Chemical-Genetic
Disruption of Clathrin does not Increase
the Colocalization of Vps33b and the
AP-1
Adaptor...........................................123-124
Supplementary Movie 1 and 2. Tridimensional
Rendering of HOPS Subunit Vps41, AP-3,
and Clathrin Localization to Enlarged
Early Endosomal Compartments....................125
Supplementary Movie 3-6. In Vivo Chemical-Genetic
Disruption of Clathrin Chains Rapidly
Redistributes
Vps18.......................................125
Chapter III.
Discussion.............................................................126-146
A Novel Model of Class B and C Vps HOPS Complex
Subunit Localization in Mammalian Cells: Overview
of Findings and
Significance............................................127-130
Question #1: Could HOPS Complex Subunits Interact
with Alternate
Adaptors?.................................................130-131
Question #2: Are HOPS Subunits Involved in Vesicle
Cargo Recruitment During Vesicle
Formation?................131-132
Question #3: Are HOPS Subunits Involved in
Cargo Sorting During Endosome
Maturation?.................132-133
Question #4: Can My Novel Model of HOPS
Localization Give Insight Into CORVET
Localization and Trafficking
Mechanisms?......................133-134
Question #5: Might HOPS Complex Have a Role in
Lysosome-Related Organelle Fusion Events, and
Polarized Mammalian Cells That Cannot Be
Recognized in
Yeast?.......................................................134-137
Question #6: Can My Novel Model of Clathrin-
Dependant HOPS Localization Explain How
Defects in HOPS Genes Contribute to
Lysosomal Human
Diseases?...........................................137-138
Summary.........................................................................139-140
Figures.............................................................................141-146
Figure 1. Model of Vesicle
Transport.............................141
Figure 2. Canonical Model of HOPS
Complex
Localization....................................................142
Figure 3. Novel Model Of HOPS Complex
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