Table Of ContentMembrane Biogenesis
NATO ASI Series
Advanced Science Institutes Series
A series presenting the results of activities sponsored by the NA TO Science
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H Cell Biology
Series H: Cell Biology Vol. 16
Membrane Biogenesis
Edited by
Jos A. F Op den Kamp
Laboratory of Biochemistry
State University of Utrecht
Padualaan 8, P.O. Box 80054
3508 TB Utrecht, The Netherlands
Springer -Verlag
Berlin Heidelberg New York London Paris Tokyo
Published in cooperation with NATO Scientific Affairs Division
Proceedings of the NATO Advanced Study Institute on New Perspectives in the
Dynamics of Assembly of Biomembranes held in Cargese, Corsica, France,
August 24 - September 4, 1987
ISBN-13: 978-3-642-73186-0 e-ISBN-13:978-3-642-73184-6
001: 10.1007/978-3-642-73184-6
Library of Congress Cataloging-in-Publication Data. Nato Advanced Study Institute on New Perspectives in
the Dynamics of Assembly of Biomembranes (1987: Cargese, Corsica) Membrane biogenesis 1e dited by Jos
A. F. Op den Kamp. p. cm.-(NATO ASI series. Series H, Cell biology; vol. 16) "Proceedings olthe NATO
Advanced Study Institute on New Perspectives in the Dynamics of Assembly of Biomembranes held in
Cargese, Corsica, France, August 24- September 4, 1987"-Tp. verso. "Published in cooperation with NATO
Scientific Affairs Division." Includes index.
ISBN-13:978-3-642-73186-0 (U.S.)
1. Membranes (Biology)-Congresses. I. Kamp, Jos A. F. Op den (Jos Amoldus Franciscus), 1939-. II. North
Atlantic Treaty Organization. Scientific Affairs Division. III. Title. IV. Series. [DNLM: 1. Biogenesis-congres-
ses. 2. Cell Membrane-congresses. 3. Membrane Lipids-congresses. QH 601 N2797n 1987]
QH601.N377 1987 574.87'5-dc 19 DNLMIDLC for Library of Congress 88-4471
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© Springer-Verlag Berlin Heidelberg 1988
Soltcover reprint of the hardcover 1st edrtion 1988
2131/3140-543210
PREFACE
Many individual aspects of the dynamics and assembly of biological membranes have been studied
in great detail. Cell biological approaches, advanced genetics, biophysics and biochemistry have
greatly contributed to an increase in our knowledge in this field.lt is obvious however, that the
three major membrane constituents - lipids, proteins and carbohydrates- are studied, in most cases
separately and that a coherent overview of the various aspects of membrane biogenesis is not
readily available.
The NATO Advanced Study Institute on "New Perspectives in the Dynamics of Assembly of
Biomembranes" intended to provide such an overview: it was set up to teach students and
specialists the achievements obtained in the various research areas and to try and integrate the
numerous aspects of membrane assembly into a coherent framework. The articles in here reflect
this. Statting with detailed contributions on phospholipid structure, dynamics, organization and
biogenesis, an up to date overview of the basic, lipidic backbone of biomembranes is given.
Extensive progress is made in the research on membrane protein biosynthesis. In particular the
post- and co-translational modification processes of proteins, the mechanisms of protein
translocation and the sorting mechanisms which are necessary to direct proteins to their final, intra
- or extracellular destination have been characterized in detail. Modern genetic approaches were
indispensable in this research area: gene cloning, hybrid protein construction, site directed
mutagenesis and sequencing techniques elucidated many functional aspects of specific nucleic acid
and amino acid sequences.
Sandwiched inbetween these articles dealing specifically with lipids or proteins are those
contributions in which the coherence and mutual interactions of membrane constituents are most
clearly expressed. The fact that many different interactions, including covalent bonding, between
lipids, carbohydrates and proteins do occur as well as the increasing awareness of the effects that
the membrane components can exert on each other with respect to structure, localization, mobilty
and function have been clearly described
Utrecht, November 1987
Jos A.F. Op den Kamp
CONTENTS
Techniques to determine trans bilayer organization and dynamics of membrane
phospholipids.
B.Roelofsen and J.AF.Op den Kamp
Transbilayer organization and mobility of phospholipids in normal and pathologic
erythrocytes.
L.L.M.van Deenen, B.Roelofsen and J.AF.Op den Kamp 15
Lipid localization and mobility in the plasmalemma of the aortic endothelial cells are
reversibly affected by the presence of cell junctions.
J.F.Tournier, ALopez, N.Gas and J.F.Tocanne 29
Phospholipid dynamics in membrane biogenesis in hepatocytes.
J.AHiggins 37
The regulation of phosphatidylcholine synthesis at the subcellular level in Krebs II
ascite cells.
F.Terce, M.Record, G.Ribbes, H.Chap and L.Douste-Blazy 59
Phospholipid asymmetry during erythropoiesis.
J.AF.Op den Kamp, ARawyler, P.van der Schaft and B.Roelofsen 67
Factors which may alter the assembly of biomembranes so as to influence their
structure or function.
F.A.Kummerow 85
Myocardial cell death and the possible role of sarcolemmal phospholipids (based on
morphological observations).
J.APost, TJ.C.Ruigrok, J.M.J.Lamers, P.D.Verdouw and AJ.Verkleij 103
The role of mitochondrial membrane phospholipid polar headgroups in yeast
cytochrome c oxidase kinetics.
ATrivedi and E.R.Tustanoff 119
The regulation of bilayer stability in bacteria.
H.Goldfine 129
Membrane structure and dynamics by NMR. Part I: effect of cyclopropane ring,
double bonds and sterols on the structure and dynamics of phospholipid membranes.
E.J.Dufourc 141
Membrane structure and dynamics by NMR. Part II: the action of polyene
antibiotics and toxins on biological membranes
E.J.Dufourc 177
Interaction of the bibenzimidazole derivative Hoechst 33258 with lipid bilayers
- a fluorescence study.
M.R.Rajeswari and G.S.Singhal 201
Physical membranes: a short review.
D.Somette 207
Anchoring of membrane proteins by glycosyl-phosphatidylinositol.
M.G.Low 219
VIII
Fatty acylation of proteins.
M.F.G.Schmidt and M.Schmidt 235
Interaction of the mitochondrial precursor protein apocytochrome c with
modelmembranes and its implications for protein translocation.
B.de Kruijff, ARietveld, W.Jordi, T.A.Berkhout, R.A Demel, H.Gorissen
and D.Marsh 257
The LDL receptor: structural insight from human mutations.
W.J.Schneider 271
The assembly and transfer of oligosaccharide chains to proteins.
W.J.Lennarz 287
Sequence determinants of protein sorting into and across membranes.
G.von Heijne 307
Components involved in protein translocation across the membrane of the
endoplasmic reticulum.
B.Dobberstein 323
Import of small secretory and plasma membrane proteins into the endoplasmic
reticulum.
R.Zimmerman, M.Sagstetter, G.Schlenstedt, H.Wiech, B.Kasseckert and
G.Muller 337
Biogenesis and membrane topology of outer membrane proteins in Escherichia coli.
J.Tommassen 351
What can we learn from colicins about the dynamics of insertion and transfer of
proteins into and across membranes.
C.J.Lazdunski 375
Bacterial protein translocation.
E.Crooke and W.Wickner 395
The use of hybrid proteins in the study of protein targeting signals.
AP.Pugsley 399
Pullulanase: a new specific secretion pathway in Escherichia coli.
C.d'Enfert, I.Reyss, ARyter and AP.Pugsley 419
A genetic analysis of pullulanase export from Klebsiella aerogenes.
M.G.Komacker, ABoyd and G.S.Plastow 429
Cloning of xcp genes possibly involved in protein secretion in
Pseudonwnas aeruginosa.
M.Bally, AFilloux, M.Murgier, B.Wretlind and ALazdunski 439
Vacuole division and inheritance in Saccharomyces cerevisiae.
B.Guthrie and W.Wickner 445
The lactose carrier of E.coli: : protease dependent in vivo inactivation.
E.Shechter,C.Houssin, A.Ghazi and H.Therisod 451
Subject Index 471
TECHNIQUES TO DETERMINE TRANSBILA YER ORGANIZATION AND
DYNAMICS OF MEMBRANE PHOSPHOLIPIDS
Ben Roelofsen and Jos A.F. Op den Kamp
Department of Biochemistry,
University of Utrecht,
P.O. Box 80.054,
3508 TB Utrecht,
The Netherlands
INTRODUCTION
Nowadays, there are no more arguments opposing the view that the structural backbone of
a biological membrane is provided by a lipid bilayer, the fatty acyl chains of the
phospholipids forming the hydrophobic core of the membrane and their polar head groups
facing the aqueous environments at either side of the bilayer. As a consequence of the
primary task of a membrane, which is to separate two aqueous compartments from one
another in which entirely different processes take place, it is conceivable that the chemical
characteristics of one side of a membrane differ considerably from those of the other.
Indeed, the absolute asymmetry in both transversal localization and orientation of,
respectively the periferal and integral, proteins in a membrane has already been recognized
a couple of decades ago. Since the early seventies, it is also known that the different classes
of phospholipids may be distributed over both halves of the bilayer in a highly asymmetric
fashion, which phenomenon particularly applies to plasma membranes. The first
information on this point has been gained from studies on erythrocytes and, still, the
membrane of those cells is the best characterized one with regards to the trans bilayer
organization and dynamics of its lipids (Op den Kamp, 1979).
NATO AS] Series, Vol. H16
Membrane Biogenesis, Edited by J.A.F. Op den Kamp
© Springer-Verlag Berlin Heidelberg 1988
2
TECHNIQUES FOR PHOSPHOLIPID LOCALIZATION STUDIES
The three types of probes that are most commonly used to assess the trans bilayer
distribution of phospholipids in a membrane are: (i) group specific chemical reagents, (ii)
phospholipases and (iii) phospholipid transfer proteins. The general strategy for their
application in such studies is simple and straightforward. Intact, closed membranes are
exposed to the action of the probe and the modification of the phospholipids thus obtained
is compared with that after treatment of open membranes with the same reagent. The
philosophy behind this approach will be obvious. When intact cells are treated with the
reagent, only those phospholipids will be modified that are localized in the outer half of the
bilayer. Treatment of open membranes serves as an essential control to make sure that,
under the cQnditions applied, the probe can react with all of its potential target molecules
when it has access to both sides of the membrane.
This general strategy implies a number of prerequisites which should be fulfilled to achieve
conclusive results. These comprise the following: (i) the structural integrity of the
membrane should be maintained during the experiment, so that the action of the probe
remains restricted to that side of the membrane where it is supposed to explore its action;
(ii) this also implies that, preferably, the membrane studied should be impermeable to the
reagent; (iii) trans bilayer movements of phospholipids should be absent, or at least very
slow relative to the time scale of the experiment; and (iv) the treatment as such should not
give rise to trans bilayer rearrangements of phospholipids.
Each of the three above mentioned types of probes will be briefly discussed below.
Group specific chemical reagents
In the past, quite a number of chemical reagents have been developed which specifically
react with free NH2-groups (Op den Kamp, 1979; Etemadi, 1980), and therefore can
provide information as to the localization of the amino-phospholipids,
phosphatidylethanolamine (PE) and phosphatidylserine (PS). A reagent that has been most
widely used for this purpose is tri-nitrobenzenesulphonic acid (TNBS). However, the
reaction between TNBS and amino-phospholipids may be incomplete, particularly in case
of a membrane containing relatively high amounts of these phospholipids, because of
sterical hindrance as a consequence of the introduction of the bulky TNB-group, whereas
the reactivity of the negatively charged TNBS towards PS will be impaired by electrostatic
repulsion between this reagent and the -also negatively charged- polar head group of this
phospholipid (Op den Kamp, 1979). Moverover, and in contrast to what has been believed
until recently, TNBS can easily permeate through the red cell membrane (Haest et al.,
1981).
Another NH2-group specific reagent is fluorescamine, which has some major advantages.
3
When applied at pH 8.0 (or higher), it reacts with free NH2-groups within a second, the
excess of the reagent being destroyed by aqueous hydrolysis within a minute. In contrast to
this hydrolysis product, the product fonned by reaction with a NH2-grouP is a fluorophor,
which enables a sensitive quantitative determination of the reaction product. That this
probe, despite its ability to permeate through a membrane, can be satisfactorily used to
determine the distribution of PE in the plasma membrane of a cell which also contains
intracellular membrane systems, is shown in figure 1.
1~
60
40
20
o 1 234 5 6 7 8
FLUORESCAMINE ADDED (,umol<zo/60x106c<zlls)
FIGURE 1
Semi-logarithmic plot of the loss in phosphatidylethanolamine (0) and of the appearance of its
fluorescamine derivative (0) in Friend cells labeled at 0-4"C with increasing amounts of
fluorescamine. Phosphatidylethanolamine pools attributable to the outer and inner plasma
membrane monolayer and to the intracellular membranes were derived by extrapolating lines a and
b, resp., of the curve representing the loss of phosphatidylethanolamine (0).
Intact Friend erythroleukaemic cells have been exposed to increasing concentrations of
fluorescamine. The semi-logarithmic dose-response curves of the labeled, respectively
unlabeled, PE in these treated cells, reveals three different pools (Rawyler et al., 1984).
The first and fast reacting pool represents the PE in the outer leaflet of the plasma
membrane, the second pool represents that fraction of the PE in the inner monolayer of this
membrane, and the third and slowly reacting pool reflects the PE located in the subcellular
membranes. The distribution of PE in the plasma membrane of these cells, as determined
by this technique, could be confinned by experiments using phospholipases (Rawyler et
al., 1985).
Plwsplwlipases
Phospholipases represent the most popular group of probes used in phospholipid
