Targeting of the Lysosomal Tether, The HOPS Complex, Open Access

Zlatic, Stephanic (2011)

Permanent URL: https://etd.library.emory.edu/concern/etds/p2676v83c?locale=en
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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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