Golgi Apparatus: Structure, Functions and Mechanisms

180 Pages·2011·3.06 MB·English

by  Christopher J. Hawkins

CELL BIOLOGY RESEARCH PROGRESS

G A :
OLGI PPARATUS
S , F
TRUCTURE UNCTIONS
M
AND ECHANISMS

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CELL BIOLOGY RESEARCH PROGRESS

G A :
OLGI PPARATUS
S , F
TRUCTURE UNCTIONS
AND MECHANISMS

CHRISTOPHER J. HAWKINS
EDITOR

Nova Science Publishers, Inc.
New York
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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA
Golgi apparatus : structure, functions and mechanisms / editor, Christopher
J. Hawkins.
p. cm.
Includes index.
ISBN 978-1-61122-611-9 (eBook)_
1. Golgi apparatus. I. Hawkins, Christopher J.
QH603.G6G656 2010
571.6'56--dc22
2010037903

Published by Nova Science Publishers, Inc. + New York
CONTENTS

Preface vii
Chapter 1 Golgi Organization and Stress Sensing 1
Suchismita Chandran and Carolyn Machamer
Chapter 2 Functional Relationships Between Golgi Dynamics and Lipid
Metabolism and Transport 43
Asako Goto and Neale Ridgway
Chapter 3 Signaling Pathways Controlling Mitotic Golgi Breakdown in
Mammalian Cells 91
Inmaculada López-Sánchez and Pedro A. Lazo
Chapter 4 Golgi Apparatus and Hypericin-Mediated
Photodynamic Action 105
Chuanshan Xu, Xinshu Xia, Hongwei Zhang
and Albert Wingnang Leung
Chapter 5 Role of the Trans-Golgi Network (TGN) in the Sorting of
Nonenzymic Lysosomal Proteins 117
Maryssa Canuel, Libin Yuan and Carlos R. Morales
Chapter 6 Golgi Apparatus Functions in Manganese Homeostasis
and Detoxification 151
Richard Ortega and Asunción Carmona
Index 161

PREFACE

The Golgi apparatus is an organelle found in most eukaryotic cells. The primary function
of the Golgi apparatus is to process and package macromolecules, such as proteins and lipids,
after their synthesis and before they make their way to their destination. This book presents
topical research data in the study of Golgi apparatus, including Golgi organization and stress
sensing; signaling pathways controlling mitotic Golgi breakdown in mammalian cells; the
role of Golgi apparatus in the biological mechanisms of hypericin-mediated photodynamic
therapy; the role of the Trans-Golgi Network (TGN) in the sorting of nonenzymic lysosomal
proteins; and the mechanisms involving the role of Golgi apparatus alteration in neurological
disorders triggered by manganese.
Chapter 1 - The eukaryotic Golgi complex plays a central role in processing and sorting
of cargo in the secretory pathway. The mammalian Golgi apparatus is composed of multiple
stacks of cisternal membranes that are organized laterally into a ribbon-like structure at a
juxtanuclear location. The stacks are polarized and protein cargo moves through the organelle
in a cis-to-trans direction. In addition, trans-Golgi membranes come in close apposition with
specialized endoplasmic reticulum (ER) membranes. These contacts are believed to mediate
lipid transfer from the ER directly to the trans-Golgi. The Golgi ribbon structure is unique to
vertebrate cells as lower eukaryotic cells lack this elaborate architecture. The complexity of
the Golgi ribbon is intriguing and suggests potential additional functions. In this chapter, we
discuss the structure of the mammalian Golgi ribbon and its potential role as a sensor of
cellular stress. We focus on the role of Golgi organization in ceramide trafficking. Ceramide
is a potent secondary messenger in signaling and apoptosis, and its levels are tightly regulated
in cells. A protein called CERT (ceramide transfer protein) delivers ceramide from its site of
synthesis in the ER to the trans-Golgi for sphingomyelin (SM) synthesis. CERT interacts with
both ER and Golgi membranes, and may function at the ER-trans-Golgi contact sites. Some
Golgi structural perturbations reduce SM synthesis as well as CERT‟s colocalization with
Golgi markers, suggesting that the organization of the mammalian Golgi ribbon together with
CERT may promote specific ER-Golgi interactions for efficient delivery of ceramide for SM
synthesis. Under cellular stress, caspase activation can lead to Golgi ribbon disassembly and
loss of ER-trans-Golgi contact sites. Prolonged stress that cannot be repaired usually results
in apoptosis. Interestingly, increased ceramide levels have been associated with apoptosis, but
it is not yet known if newly synthesized ceramide resulting from perturbation of ER-trans-
Golgi contact sites contributes to ceramide signaling during apoptosis. An important question
is whether ER-trans-Golgi contact sites are upstream targets of stress signals leading to
increased ceramide levels and caspase activation, or if altered ceramide trafficking is
viii Christopher J. Hawkins
downstream of Golgi disassembly. Regardless, it is clear that Golgi ribbon structure,
including ER-trans-Golgi contact sites is exquisitely sensitive to perturbation, making this
organelle an ideal platform to sense cellular stress and integrate signals that determine cell
survival or cell death.
Chapter 2 - The Golgi apparatus is a sorting nexus for protein and lipids exported from
the endoplasmic reticulum (ER) to other organelles and for secretion. The lipids and sterols
that delineate the vesicular/tubular transport carriers and cisternae that constitute the Golgi
transport apparatus are just packaging materials but participate directly in membrane fusion,
cargo sorting and polarized transport. Low abundance lipids, such as diacylglycerol (DAG),
phosphatidic acid (PtdOH), lyso-phospholipids and phosphatidylinositol phosphates,
contribute to these processes by localized synthesis and interconversion. These lipids alter the
structure of membranes by assisting in induction of positive and negative curvature required
for carrier assembly, and regulate the activity of proteins that temporally and spatially
regulate fusion and fission events. The Golgi apparatus is especially enriched in
phosphatidylinositol 4-phosphate (PtdIns(4)P), where localized metabolism by Golgi-
associated PtdIns 4-kinases (PI4K) and phosphatases controls PtdIns(4)P pools that recruit
proteins involved in lipid transport and vesicular trafficking. DAG and ceramide conversion
in the late Golgi and trans-Golgi network (TGN) by sphingomyelin (SM) synthase regulates
Golgi trafficking by recruiting and activating protein kinase D (PKD) for phosphorylation of
targets such a PI4KIIIβ. SM and glycosphingolipids (GSL) synthesized in the Golgi apparatus
condenses with cholesterol into nanoscale assemblies called lipid rafts. These platforms
function in membrane signaling and regulate trans-Golgi network (TGN)-sorting machinery.
There is an increasing appreciation for the role of lipid and sterol transfer proteins in
modulation of Golgi apparatus function. In particular, site-directed ceramide and sterol
transfer proteins that communicate lipid status, and regulate cholesterol, SM and GSL
metabolism. Here we will review the highly integrated lipid metabolic and signaling
pathways housed in the Golgi apparatus that control secretory activity and membrane
assembly.
Chapter 3 - In mitosis, each daughter cell must receive a complete and equal set of
cellular components. Cellular organelles that are single copy, such as endoplasmic reticulum,
nuclear envelope and Golgi apparatus, have to break down to allow their correct distribution
between daughter cells. The mammalian Golgi is a continuous membranous system formed
by cistern stacks, tubules and small vesicles that are located in the perinuclear area. At the
onset of mitosis, the Golgi apparatus undergoes a sequential fragmentation that is highly
coordinated with mitotic progression and in which reversible phosphorylation plays a critical
regulatory role. In fact, several kinases have been implicated in each stage of this
fragmentation process. Before mitotic disassembly, the lateral connections between the stacks
are severed resulting in the formation of isolated cisternae. Several kinases such as mitogen-
activated protein kinase kinase 1(MEK1), Raf-1, ERK1c, ERK2, Plk3, VRK1, several Golgi
matrix proteins (GRASP65 and GRASP55) and the membrane fission protein BARS have
been shown to mediate signals in this first step that takes place in late G2 phase. As prophase
progresses, the isolated cisternae are first unstacked followed by its breakage into smaller
vesicles and tubules that accumulate around the two spindle poles at metaphase. Unstacking
and vesiculation are triggered by several proteins including kinases (Plk1 and Cdc2), the
GTPase ARF-1 and inactivation of membrane fusion complexes (VCP and NSF). Post-
mitotic Golgi reassembly consists of two processes: membrane fusion mediated by two
Preface ix
ATPases, VCP and NSF; and cistern restacking mediated by dephosphorylation of Golgi
matrix proteins (GRASP65 and GM130) by phosphatase PP2A (Bα). Apart from the tight
regulation by reversible phosphorylation, it seems that mitotic Golgi membrane dynamics
also involves a cycle of ubiquitination during disassembly and deubiquitination during
reassembly in part regulated by the VCP-mediated pathway.
Chapter 4 - Hypericin isolated from Hypericum perforatum plants, exhibits a wide range
of biological activities and medical applications for treating tumors. Emerging evidence has
demonstrated that hypericin could be activated by visible light to produce reactive oxygen
species (ROS) which destroys a tumor directly or indirectly. Hypercin-induced photodynamic
therapy has showed considerable promise as an alternative modality in the management of
malignant tumors. However, the exact mechanisms need to be clarified. Recent studies have
showed that hypericin was accumulated in the Golgi apparatus, indicating that the role of the
Golgi apparatus is indispensable in the biological mechanisms of hypericin-mediated
photodynamic therapy.
Chapter 5 - In eukaryotes the delivery of newly synthesized proteins to the extracellular
space, the plasma membrane and the endosome/lysosomal system is dependent on a series of
functionally distinct compartments, including the endoplasmic reticulum, the Golgi apparatus
and carrier vesicles. This system plays a role in the post-translational modification, sorting
and distribution of proteins to their final destination. Most cargo is sorted within, and exits
from, the trans-Golgi network (TGN). Proteins delivered to the endosomal/lysosomal system
include a large and diverse class of hydrolytic enzymes and nonenzymic activator proteins.
They are directed away from the cell surface by their binding to mannose-6-phosphate
receptors (MPR). Surprisingly, in I-cell disease, in which the MPR pathway is disrupted, the
nonenzymic sphingolipid activator proteins (SAPs), prosaposin and GM AP, continue to
2
traffic to the lysosomes. This observation led us to the discovery of a new lysosomal sorting
receptor, sortilin. Both prosaposin and GM AP are secreted or targeted to the lysosomes
2
through an interaction of specific domains with sortilin. In the case of prosaposin, deletion of
the C-terminus did not interfere with its secretion, but abolished its transport to the
lysosomes. Our investigations also showed that while the lysosomal isomer of prosaposin
(65kDa) is Endo H-sensitive, the secretory form (70kDa) is Endo H-resistant. Since the
processing pathway within the Golgi apparatus is highly ordered, this Endo-H analysis
permitted us to distinguish a sorting sub-compartment where a significant fraction of
prosaposin exits to the endosomal/lysosomal system prior to achieving full glycosylation and
Endo-H resistance. Mutational analysis revealed that the first half of the prosaposin C-
terminus (aa524-540) contains a saposin-like motif required for its binding to sortilin and its
transport to the lysosomes. Additionally, a chimeric construct consisting of albumin and a
distal segment of prosaposin, which included its C-terminus, resulted in the routing of
albumin to the lysosomes. Based on previous observations showing that the lysosomal
trafficking of prosaposin and chimeric albumin required sphingomyelin, we tested the
hypothesis that these proteins, as well as sortilin, are associated with detergent-resistant
membranes (DRMs). Our results demonstrated that indeed sortilin, prosaposin and chimeric
albumin are found in DRMs, and that the sorting of prosaposin to DRMs depends upon the
interaction of its C-terminus with sortilin. In conclusion, we have identified a specific
segment in the C-terminus of prosaposin, as well as amino acid residues that are critical to the
binding of prosaposin to sortilin and its subsequent lysosomal trafficking. The identified