On the microassembly of integral membrane proteins.

ANNUAL REVIEWS Annu. Rev. Biophys. Biophys. Chern. 1990. 19:369..-403 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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ON THE MICROASSEMBLY OF INTEGRAL MEMBRANE PROTEINS! Jean-Luc Popot and Catherine de Vitry

Serv ice de Photosynthese, Institut de Biologie Physico-Chimique, rue Pierre-et-Marie Curie, F-75005 Paris, France

13

KEY WORDS:

protein traffic, folding domains, organelle membrane proteins, integral membrane protein subunits, protein import into chloro plast and mitochondrion.

CONTENTS PERSPECTIVES AND OVERVIEW ................ ... ................ .. .......... ......... ......... ............. ..........

Identifying Putative Transmembrane Segments and Estimating Their Hydrophobicity

370 371 371 373 3 74 374

Number and IIydrophobicity of Putative Transmembrane a-Helices as a Function of Protein Localization and Function ................. ... .. ...... ............... . . ..................

382

PROCEDURES.................................................................................................................

Hydrophobicity Analysis . . . . . . . . . . . .. .. .... .... . . . . . . . . . . .. . . . . .. ....... . . . . . . . . . . . . . . . . . . . . .. ......... " . . . . . . . . . .

Choice of Proteins ....................... ........................... ...... ....... .....................................

RESULTS ...., .. " .. """"""""", . . . . . . ,.""""""""", .. , .. , . . . """"""""", .. , . . . """""""""", .. "",,

Number and Hydrophobicity of Transmembrane Segments in Organelle innerMembrane Proteins Depending on Site uf Synthesis..................................... DISCUSSION .............. . . . . . . . . . ......................... . . . . .. . . . . . . . . . . ............... ....................................

The Microassembly of Integral Membrane Proteins in Organelles ............... . .. ....... ... Other Membranes..................................................................................................... Displacing the Synthesis of Integral Membrane Proteins from Organelles fo fhe Cytoplasm .................... .... ..... .... . . .... . . . . . . . . . . . . . . . . . . . . ...............

CONCLUSION .......... ......................... . . . . . . . . . . . . . . . . . . . . . .............. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I

383 387 387 395 397 399

This review is dedicated to Annemarie Weber (University of Pennsylvania) and Andrew

G. Szent-Gyiirgyi (Brandeis University), instructors at the Physiology Summer Course at

Woods Hole, Massachusetts, in 1971.

369 0883-9 1 82/90/061 0-0369$02,00

370

POPOT & DE VITRY


PERSPECTIVES AND OVERVIEW Recent experimental evidence suggests that the folding of transmembrane regions of integral membrane proteins s hould be regarded as qualitatively different from that of soluble proteins. In soluble proteins, hierarchical levels of folding consist of the secondary structure units (a-helices, f3s trands), super-secondary motifs (e.g. the f3exf3 unit or the 8-fold exf3 barrel), domai ns (comprised of one or several motifs), protomers (comprised of one or several domains), and, if a quaternary s tructure exists, oligomers (cf 44, 1 13, 1 23). Folding is believed to follow this seq uence more or less closely, with the first independently stable structures appearing at the domain level (e.g. 39, 64, 1 44). Domains vary in size. For globular proteins, they comprise typically 70- 1 50 amino acid residues. Smaller domains , e.g. the 40-50 residue domains in rubredoxin or wheat g erm agglutin in, are found in proteins s tabilized by disulfide bridges or prosthetic' groups (for reviews, see 65, 1 1 3 , 12 3 , 1 44). As a conseq uence, soluble proteins or protein subunits smaller than'" 70 residues are very rare ( 1 29a). In the transmembrane regions of integral membrane proteins, a s ingle hydrophobic ex-helix apparently has, to a large ex ten t, the properties of a domain in itself. Both theoretical considerations and experimental obser v ations form the basis for this view (for a review, see 103). F irst, trans membrane reg ions in bacterial photos ynthetic reaction centers and in bacteriorhodopsin are composed of hydrophobic a-helices. Free energy estimates lead to the prediction that each ex-helix can form an independent, stable, transmembrane en tity in a lipid bilayer. Second, several integral membrane proteins hav e been functionally reassembled starting from frag ments that had been independently refolded or synthesized (Table 1 ) . In these experiments, each fragment folded autonomously, as expected if it is itself comprised of elements (a-helices) that can take up a largely correct transmembrane pos ition and secondary structure by themselves. In addition, two natural cases s uggest s uch a process , in which a s ingle polypeptide in on e membrane or organism appears in another to be split into two subunits (Table I). An ex-helix long enough to cross the 30-A thick fatty acyl region of a phospholipid bilayer comprises 20 residues . Transmembrane regions may thus be built up of stable units that are considerably s maller than those involved in the f olding of soluble proteins. T he possibility arises that this particularity dictates some characteristic features of membrane protein biosynthesis and assembly. In the present review, we survey the s ubunit composition, size, and number of hydrophobic transmembrane segments of most eukaryotic inte gral membrane proteins for which the sequence is known (see also 3 2a) . ,-

371

MICROASSEMBLY OF INTEGRAL MEMBRANE PROTEINS

Table 1

Integral membrane proteins assembled from fr agments that had been either refolded or biosynthesized independently

Origin of fragments


Proteolysis Proteolysis Proteolysis Proteolysis Proteolysis Engi neer ed mRNA Engineered plasmids

Natural"

Natural"

Medium where assembly occurs lipid/detergent mixed micelles lipid/detergent mixed micelles lipid vesicles lipid vesicles lipid vesicles Xenopus o ocyte (ER?) E. coli plasma memhrane thyl ak o id E.

coli plasma membrane

Protein

Hydrophobic helices per fragment

References

bacteriorhodopsin

2+5

62, 78

hacteriorhodopsin

5+2

77, 127

bacteriorhodopsin bacteriorhodopsin bacteriorhodopsin f3 2 adr energic receptor lac permease

2+5 5+2 1+ 1+5 5+2

1 04, 105 68 69 71

2+12

147

4+3

57, 145a

8+4

14g

cytochrome b, +subunit I V N i coti nami de nucleotide transhydr o gen ase

'Natural cases correspond to proteins composed of one polypeptide chain in one type of membrane or organism and two distinct subunits in another. The number of putative hydrophobic transmembrane segments in cytochrome b6, subunit IV, and nicotinamide nucleotide transhydrogenase is discussed in the footnotes to Table 4. Cytochrome h6 and subunit IV from chloroplast b61f complex are respectively homologous to the amino terminal and carboxyterminal parts of cytochrome b from the bc I complexes from mitochondria or purple bacteria; a segment homologous to the last of the 8 putative hydrophobic transmembrane a-helices in cytochrome b is missing in subunit I V. The a and {J subunits of E. coli transhydrogenase are homologous to the amino terminal and carboxyterminal parts of the beef enzyme, respectively.

This a nalysis shows that many of them indeed contain s ubunits that a re much sma ller than subunits of s oluble proteins . Very marke d differe nces in composition and properties e xist depending on which membrane the proteins lie in. A particularly striking observation is that the composition of complexes f rom the inner mitochondrial membra ne a nd thyla koid mem brane appare ntly reflects a restriction on the import of large hydroph obic proteins f rom the cytoplasm, while including a large number of very small, 1 - or 3-a-he lix s ubunits.

PROCEDURES Hydrophobicity Analysis

The seque nces of about 250 presumed or proven integral membrane pro te ins were take n from the CITI2 (Paris) data base BISANCE (data banks


372

POPOT

& DE

VITRY

NBRF, EMBL, GENPRO, and GENBANK) or collected fr om the litera ture. We examined them for the presence of potential trans membrane hydrophobic (J(-helices using a modifica tion of Klein et ai's (70) pr ogram r un on a Vax 750 computer . The progr am examined each seq uence twice. In the firs t pass, using the hydrophobicity scale of Kyte & Doolittle [KD sca le (73)] and a 1 7-residue span, the program generated the number and approximate limits of the putative transmembrane helices , together with an index of the relative probability (P/I), that each segment is either periphera l or tra nsmembrane (70). The program also gavc the average hydrophobicity of each segment (GES) expressed in kcal/residue using t he hydr ophobicity scale of Engelman et al [Table 2, GES scale (37)] . In the second pass, the search was done using the GES scale and a 1 7-residue span. The procedure is similar to that applied by von Heij ne ( 1 42a) to bacterial membrane pr otein sequences . Both searches usually identified the same hydrophobic seg ments . I n some cases, neighbor ing hydrophobic segments separ ated b y a few polar residues were properly distinguished in the fi rs t pass a nd not in the second, or vice versa . Topological models fr om the literature were then compared to r es ults from the hydrophobicity analyses as described in the section covering r esults a nd in footnotes to Table 4. For the purpose of estimating helix hydrophobicities, the limits of the s egments were defined using t he GE S scale except for a dozen cases (over about 600 segments) in which segments matching those proposed in the litera ture were better identified using the KD scale. In a ll cases and thr oughout this text, hydrophobicities are expressed as GES values . To tes t the r eliability of our approach in estima ting the hydr ophobicity of putative tra nsmembr ane helices , we a ppli ed i t to the three integ ral

Table 2

Goldman-Engelman-Steitz (GES) hydrophobicity scale

Residue

Transfer free energy"

Phenylalanine Methionine

Isoleucine Leucine Valine Cysteine Tryptophan

3.7 3.4 3.1 2.8

2.6 2 .0

1.9

Residue

Transfer free energy

Alanine

1.6

Threonine

1.2

Glycine Serine Proline Tyrosine Histidine

1.0 0.6 - 0.2 -0.7 - 3.0

Residue Glutamine Asparagine Glutamic acid Lysine Aspartic acid Arginine

Transfer free energy -4. 1 -4.8 -8 2 - 8.8 -9.2 - 1 2.3 .

'Free energy (kcal/rcsidue) for transferring residues in an �-helix from a nonaqueous environment to water (from Ref. 37).



373

membra ne proteins in the photosynthetic reaction center from purple bacteria . In all cases, the I 7-residue segments identified as the most hydro phobic using either the KD or the GES scales corresponded to the trans membrane helices. In 10 cases (out of I I helices), the segments were included within the tra nsmembrane helices of the electron density map (30a) to within 3 residues. The GES scale displaced helix E in subunit L by 7 residues past the end of the tra nsmembrane region, while the KD scale placed it correctly to within I residuc; the difference in GES value depending on which segment limits were used was 0.09 kcaljresidue. Con versely, using the KD scale resulted in misplacing helix C in subunit M by 6 residues, while the GES scale placed it correctly. The hydrophobicity difference was 0.04. Clearly, as expected, inaccuracy in defi ning helix limits entailed only small errors on GES. The transmembrane segments in the photoreaction center a re fairly hydrophobic, which simplifies identification. In bacteriorhodopsin, the 1 7residue segments identified as the most hydrophobic using either scale again matched accepted transmembrane helices (37). The least hydro phobic helix (helix C, which contains two aspartic acid residues), was predicted at the same position (within 1 residue) by the two scales. The g rea test discrepancy between the hydrophobicity estimatcs calculated using the limits given by the two scales reached 0. 1 3 (helix G). The results obtained on bacteriorhodopsin a nd the reaction center proteins are sum marized in Table 3 . Choice of Proteins

We distributed integral membrane proteins into f our sets: I. proteins from the plasma membrane of eukaryotic cells a nd of other Table 3

Number and hydrophobicity of transmembrane helices in four well-characterized integral proteins from bacterial cytoplasmic membranes GES of transmembrane segmentsa

Protein Reaction center CR. viridis): Subunit H Subunit L Subunit M Bacteriorhodopsin

A

1.63 2.60 2.31 2.02

Segment position in sequence B F C D E

1.86 2.15 2.01

2.11 2.02 0.94

1.70 1.71 1.50

1.89 1.86 2.13

1.74

G

GESavb

1.25

1.63 2.03 2.01 1.66

a Average free energy of transfer for the most hydrophobic l7-residue segment overlapping each helix (kcal/residue). b Average of the individual GES values.


374

POPOT & DE VITRY

membranes that are directly in contact with the cytosol (endoplasmic and sarcoplasmic reticulum, retina sacculae, exocytotic vesicles), which we designate as plasma membrane proteins. Several homologous proteins from the same or d ifferent species werc oftcn analyzed, although the results for only one of them are incorporated in the Figures and analysis. 2. protei ns from the mitochondrial inner membrane, whether coded f or by mitochondrial or nuclea r DNA. M itochondrial genomes were a nalyzed in totality for Bos primegenius taurus, Homo sapiens, Mus musculus, and Xenopus laevis, and par tially, as a f unction of available sequences, f or Neurospora crassa, Saccharomyces cerevisiae, Leishmania tarentolae, Try panosoma brucei, Chlamydomonas reinhardtii, and Zea mays. We also analyzed the proteins of the mitochondrial inner membrane of these species encoded by nuclear DNA and of known sequence. The results are s hown for bovine proteins except if the sequence was only available for other eukaryotes. The two hydrophobic s ubunits of the succinate dehydrogenase complex have similar molecular weights in eukaryotes and in the pro karyote Escherichia coli and, to our k nowledge, have not been sequenced in any eukaryotes . In this particular case, we show the results for E. coli. 3. protei ns f rom the thylak oid membra ne, whet her encoded in the chlo roplast or the nucleus, and chloroplast DNA open reading frames (ORF s) encoding putative, unidentified proteins . The chloroplast gcnomcs of Mar chantia polymorpha and Nicotiana tabacum were analy zed in totality, although only the results for Marchantia are shown, except when the subuni ts were better characterized in Nicotiana. The integral pr oteins of the thylakoid membra nes encoded in the nucleus were analyzed in several higher plants as well as in C. reinhardtii, as a function of available sequences . 4. proteins from the outer membran e of E. coli. When several homologous proteins from d ifferent organisms or repre senting rela ted enzymes were exa mined , only one protein of each family was included in the fi na l a na lysis unless sequence similarities were less tha n 35--40%. The alignment program (alignment score program of B. C. Orcutt, M. O. Dayhoff, and W . C. Barker , 1984 v ersion available at the CITI2) was based on the a lgorithm of Needleman & Wunsch (89) using a unitary matrix and a break penalty parameter of 8.

RESULTS Identifying Putative Transmembrane Segments and Estimating Their Hydrophobicity

Table 4 lis ts 140 integra l membrane proteins, which covers nearly all eukaryotic integral membrane proteins of known sequence when proteins


Table 4

375

Number and hydrophobicity of putative hydrophobic transmembrane segments in

integral proteins from eukaryotic membranes"

4A

Proteins from membranes that are directly in contact with the cytosol {plasma


membrane, endoplasmic and sarcoplasmic reticulum, retina sacculae, exocytotic vesic1es)h

Protein

Genus

cUI ON. A (DZa) gene product class I "37" protein T-cell L3T4 glycoprotein T-cell CD3 glycoprotein Il chain T-cell receptor �-chain precursor B-lymphocyte glycoprotein PCI

Homo Mus Mus Homo Oryctolagus Homo Mus Rattus Homo Gallus Gallus Homo Homo Homo Mus Homo Drosophila Homo Homo Homo Homo Homo Homo Drosophila Caenorhabditis Drosophila Homo Homo Rattus Rattus Rattus Rattus Homo Rattus Arbacia Oryctolagus Bos Rattus Bos Rattus Cricetulus Rattus Bos Bos Drosophila Homo Cricetulus Rattus

HLA

H2

Uvomorulin Asial oglyc oprotein receptor

Fibronectin receptor a chain Fibronectin receptor � chain N-CAM G1ycophorin A GIycophorin c! ILGF-II receptor POGF receptor EGF receptor EGF receptor NGF receptor Lymphocyte IgE receptor

Interleukin-2 receptor P55 chain

Insulin recep tor Transferrin receptor

LOL receptor Toll gene product lin-12 gene product Notch gene product Thy-1 antigen CSF-1 receptor (c-fms) High affinity IgE rec. a chain High affinity IgE rec. � chain' High affinity 19E rec. y chain �-gal. CI. 2,6-sialyltransferase Aminopeptidase N Enkephalinase Guanylate cyclase Intestinal sucrase-isomaltase Cytochrome bs Stearyl-CoA desaturase Cytochrome P-450 (C21) NADPH-cyt. P-450 oxidoreductase HMG-CoA reductase Synaptophysin Myelin proteolipid Opsin Op sin

M2 muscarinic receptor �2 adrenergic receptor O2 dopaminergic receptor

N,

Nh

225 337 435 150 (319) 905

1 1 1 1 1 1 1 1 1

728 284

1008 803 1072 131 128

2451 1067 1I86 1330 399 321 251 (1370) 760 860 (1097) 1429 (2703) 142 (959) 222 243 62

403 967 750 955 (1827) 135 358 496 678 887 307 276 348 373 466 418 415

I

1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 4 1 1 1 1 1 1 1 (4) (1) 1 7 4

(4) 7 7 7 7

7

min

GES avo max. Ref.

2.42 2.49 2.32 1.99 2.11 2.23 2.62 2.42 2.66 2.31 2.62 2.47 2.52 2.33 2.59 2.42 2.37 2.07 2.18

2.27

2.49

2.46 2.57 2.33 2.54 2.56 1.69 2.57

NBRF 73a NBRF NBRF

NBRF NBRF 114 NBRF EMBL 134 29 NBRF NBRF

84 149 NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF 54 150 NBRF NBRF 25

1.57 10 1.75 2.18 2.55 10 1.51 10 2.71 Gpro 2.28 94 2.39 Gpro 2.01 129 2.77 Gbk NBRF 1.78 1.32 1.78 2.04 136 1.91 NBRF 2.41 NBRF 0.68 1.58 2.29 NBRF 1.70 1.97 2.27 75 1.99 2.17 2.29 NBRF 1.41 2.00 2.38 NBRF 0.70 1.69 2.06 NBRF 0.63 1 .7 5 2.36 99 0.79 1.83 2.54 NBRF 1.13 1.90 2.57 16 continued

376

POPOT & DE VITRY

Table 4

(continued)

Protein

Genus

1c serotonin receptor a-factor receptor (S1E2) a-factor receptor (S1E3) Substance K receptor mas oncogene S Ll hCG receptor Ca ATPase (slow twitch muscle) z Ca ATPase (plasma membrane) Na+/K+ ATPase a chain

t


:

Na+/K+ ATPase � chain + Na /K+ ATPase putative y chain* H AJPase (plasma membrane) H /K ATPase

:

Adenylyl cyclase$ Uracil transport protein Gi,:!cose transporter Na Iglucose co-transporter Arginine permease P-glycoprotein patched gene product Anion exchange protein Nicotinic ACh receptor a chain Glycine receptor 48K chain GABAA receptor ex ch ain Voltage gated Na+ channel cl+ channel (XI subunit K+ channel (Shaker gene) Ryanodine receptor $ Inositol trisphosphate receptor Lens fiber MP26 Gap junction connexin

(mdrl\

4B

Rattus Saccharomyces S accharomyces Bos H omo Sus Oryctolagus Homo Ovis Gal/us 'Ovis Neurospora Rattus Bos S accharomyces

Homo Oryctolagus S accharomyces Homo Drosophila Mus Torpedo Rattus Bos Electrophorus Oryctolagus Drosophila Oryctolagus Mus Bos Rattus

N, 460 431 470 384 325 674 997 1220 1021 305 68 920 1016 1134 633 492 662 590 1280 1299 929

437

421

429

1820 1873 6 16 5037 2749 263 283

Nh 7

7

7 7 7

7

10 10 (7)

1 1 (10) 7 12 12 12 (15) 7 12 12 13 4 4

4

(20) (20)

min

GE S avo

1.33

2.04

0.92 1.01 1.22 1.17 0.91 0.45 1.40

2.05 1.85 1.97 1.15 1.55 1.74 1.97

0.99

1.08 1.15 1.34 1.19 1.00 1.35 1.48 1.22 1.41 1.05 2.22 1.47

1.66

0.12 0.56

1.69

2.28 2.53 1.83 1.96 1.85 1.79 1.89 1.95 1.98 1.90 2.01 1.91 2.44 1.88

1.78

7 6 4

1.00 1.59 1.69

1.79 1.80 2.03 2.20 1.86 1.71 1.89

GE S avo

(5) 4

1.39 1.99

max.

Ref

2.75 2.13

66 NBRF NBRF Gpro NBRF

2.74

2.49 2.75 2.24 1 .99 2.42 2.42

2.57 2.44 2.66 2.56 2.75 2.58 2.37

2.59

2.43 2.59 2.64 2.09

1.97

79

NBRF 140 NBRF

Gbk 24 Gpro Gpro 72 67 Gbk 56 Gbk NBRF 88 Gbk NBRF 47

122

2.55 1.89 2.36

NBRF 135 102 133 41a Gbk Gbk

max.

Ref.

2.54 2.74 2.5 1 2.36 2.51 2.64 1.93

NBRF

2.72 2.38 2.11 2.22

NBRF

2.81 2.88 2.61

2.49

Proteins from the inner mitochondrial membranec

Mitochondrion-encoded proteins

Genus

N,

Nh

min

NADH-Q reductase, subunit 1 NADH-Q reductase. subunit 2 NADH-Q reductase, subunit 3 NADH-Q reductase, subunit 4 NADH-Q reductase, subunit 5 NADH-Q reductase, subunit 6 NADH-Q reductase, subunit 4L QH2-cyt. c reductase, cytochrome b Cyt. c oxidase, subunit I Cyt. c oxidase, subunit II Cyt. c oxidase, subunit III ATPase, subunit 6 $ ATPase, subunit 8

Bos Bos Bos Bos Bos Bos Bos Bos Bos Bos Bos Bos Bos

318 347 115 459 606 175 98 379 5 14 227 261 226 66

(8) 10

1.37

15 15 5 3 (8) 12 2 7 (5) 1

(Escherichia) (Escherichia)

128 115

(3) (3)

3

1.55

1.95 1.34 0.96 1.88 1.66 1.26 1.45 2.17 1.37 1.67

2.05 2.03 2.17 1.81 1.91 2.25 1.84 2.11 2.10 2.27 1.76

1.94

2.62

2.66

NBRF

NBRF NBRF NBRF NBRF NBRF NBRF

NBRF NBRF NBRF NBRF

Nucleus-encoded proteins Succinate-Q reductase, subunit C Succinate-Q reductase, subunit D

1.84 2.12

2.13 2.15

2.30 2.30

146 146

MICROASSEMBLY OF INTEGRAL MEMBRANE PROTEINS Table 4 (continued) Genus


Nucleus -encoded proteins

Bos QH2-cyt. c reductase. cytochrome c1 Bos QH2-cyt. c reductase. FeS proteinO Bos QH2-cyt. c reductase. subunit VIf Bos QH2-cyt. c reductase. subunit X# Bas QH2-cyt. c reductase. subunit XI# Bas Cytochrome c oxidase. subunit IV Cytochrome c oxidase. subunit VIIIl Bos Cytochrome c oxidase. subunit VIIIb# Bos Bos ATPase. subunit 9 S accharomyces Threonine deshydrataseo Bas Nicotinamide nucl. transhydrogenaseO Bos ADP/ATP carrier proteino Cricetulus Brown fat uncoupling proteino Bos Phosphate carrier proteinO

4C

min

N,

Nh

241 196 81 62 56 147 47 46 75 576 1043 297 306 313

1 1 1 1 1 1 1 2 1 (12) (3) 3 3

1.51 1.13 1.41 0.68

N,

Nh

min

368 501 120 499 692 191 100 343 508 459 353 83 39 73 36 40 37 38 285 215 160 750 184 81 248

(7) 15 3 14 17 5 3 5 6 6 5 1 1 1 1 1 1 1 I 4 3 11 1 2 (5)

1.54 1.28 1.38 1.27 1.13 1.61 0.91 1.15 1.48 1.60 1.91

179 87 233 202 (179) (241)

1 1 3 3 3 3

I

1.86

377

GE S avo max. Ref

1.69 1.51 1.22 0.96 1.44 2.49 2.39 2.04 2.28 1.92 1.74 1.45 1.61 1.53

2.71

1.97 1.76 1.96 2.12

NBRF 120 11 NBRF 119 NBRF NBRF NBRF NBRF NBRF 148 NBRF NBRF NBRF

Proteins from the thylakoid membraned

Chloroplas t-encoded proteins

NADH-Q reductase. subunit NADH-Q reductase. subunit NADH-Q reductase. subunit NADH-Q reductase. subunit NADH-Q reductase, subunit NAOH-Q reductase, subunit NADH-Q reductase, subunit psn, subunit 01 (psbA) psn. subunit 47kDa (psbB) psn, subunit 44kDa (psbC) psn, subunit 02 (psbD)

Genus

1 2 3 4 5 6 4L

Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Marchantia Nicoliana Nicotiana psn. phosphosubunit (ps bH) Nicotiana psn, subunit encoded by psbt Marchanlia psn, subunit encoded by psb! Marchantia psn, subunit encoded by psb[(l Nicotiana # psn, subunit encoded by psbL Marchantia Cytochrome brlt. cytochrome t (petA) Marchantia Cytochrome brlt. cytochrome b6 (petB) Marchantia Cytochrome brit, subunit IV (petD) Marchantia PSI. subunit P700 (psaAJ) Marchantia ATPase, subunit I (atpF) Marchantia ATPase. subunit TIl (atpH) Marchantia ATPase, subunit IV (atpi) Marchantia

��: ��::�� : ��:

G ES avo max. Ref

1.74 1.71

1.93 1.90 2.07 1.85 2.04 2.03 1.68 1.82 1.97 1.97 2.11 1.99 1.82 2.54 2.54 2.35 2.18 2.24 2.26 1.94 2.13 1.71 0.94 1.83 2.01

1.22 1.19 0.94 1.13

1.77 131 1.74 41 1.66 2.39 17 1.39 1.62 61 1.10 1.41 130 1.41 1.78 101

1.39 2.11 1.13

2.63 2.56 2.79 2.42 2.69 2.46 2.45 2.27 2.24 2.45 2.29

2.39 2.19 2.35 1.92 2.30

NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF Gpro Gpro Gpro NBRF NBRF 86 NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF

Nucleus-encoded proteins

Cytochrome briJ. Rieske FeS proteinO PSI. subunit P37# LHCII chlo. ab protein type 1° LHCI chlo. ab protein type 1° LHCI chlo. ab protein type IIo LHCI chlo. ab protein type IIIo

S pinacia Chlamydomonas Pisum Lycopersicon Petunia Lycopersicon

continued

378


4D

POPOT & DE VITRY

Protein from the inner envelope membrane of chloroplastd GES

Protein

Genus

Phosphate translocator"S

Spinacia

4E

NT

Nh

min

avo

max.

Ref.

(404)

7

1.24

1.75

2.05

40

Hypothetical membrane proteins encoded by chloroplast open reading frames (ORFs)d

Protein Hypothetical protein 135 Hypothetical protein 184 Hypothetical protein 1068 Hypothetical protein 203 Hypothetical protein 288 Hypothetical protein 2136

Hypothetical pTotein 320

# Hypothetical protein 36b Hypothetical protein 434 # Hypothetical protein 42� Hypothetical protein 31 # Hypothetical protein 32 # Hypothetical protein 33 # Hypothetical protein 34 # Hypothetical proteirt 35 ' Hypothetical protein 37 # Hypothetical protein 50 # Hypothetical protein 55 Hypothetical protein 62 Hypothetical proteirt mbpX

Genus Marchantia Marchantia Marchantia Marchantia Marchanlia Marchantia Marchantia Marchanlia Marchantia Marchantia Marchanlia Marchantia Marchantia Marchantia Marchanlia Marchantia Marchantia Marchantia Marchantia Marchantia

NT

Nh

min

135 184 1068 203 288 2136 320 36 434 42 31 32 33 34 35 37 50 55 62 370

3 2 6 1 6 2 6 1 5 1 1 1 1

2.02 2.17 1.36 1.83 1.78 1.32 1.32

1

1 1 1 1 2 2

2.21 1.22

GES avo max.

2.13 2.42 1.84 2.01 2.08 1.94 1.88 2.16 1.66 2.17 2.54 2.09 2.11 2.45 2.64 2.24 2.16 2.18 2.35 1.29

2.22 2.67 2.11 2.59 2.10 2.24 1.91

Ref NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF

NBRF

2.48 1.36

NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF NBRF

" N, is the number of residues in the mature protein, except in a few cases as indicated by parentheses. These cases are discussed in Footnotes lr- r

; ;�

> Z tTl

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o

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Table 6

Comparison of integral membrane proteins from eukaryotic organelles and from prokaryotesa 6A

Mitochondrion vs prokaryote Eukaryote & prokaryote

Eukaryote specific Prokaryote


Complex

Mito. Nuc!. Milo. Nuc!.

NADH-Q reductase Bos touris (vs E. coli)

specific

151 8 10 3

Subunit 1 Subunit 2 Subunit 3 Subunit 4 Subunit 5 Subunit 6 Subunit 4L

IS

15 5 3

Only one subunit (47 kDa, o helix)

55, 1 46

Succinate-Q reductase

Bus tauris (vs E. culi) Subunit C

(3) (3)

Subunit D

QH2-Cytochrome c Reductase Bas touris (vs Rhudobocter sphoeroides) Cytochrome b Cytochrome c , FeS protein Subunit VII Subunit X Subunit XI Cytochrome c oxidase Bas tauris (vs Paracoccus denitri/icans) Subunit I Subunit II Subunit III Subunit IV Subunit VIIIa Subunit VIIlb

Reference

-

42 8

108 12 2 7

ATPase

Bas tauris (vs Subunit 6 Subunit 8 Subunit 9

81

E. coli)

Nicotinamide nucleotide transhydrogenase Bas tauris (vs E. coli subunits 0: and (J)

Subunit b (1 helix)

5 2

12

(4 + 8)

22

MTCROASSEMBLY OF INTEGRAL

6B

Chloroplast vs prokaryote Eukaryote & prokaryote

Complex


39 1

MEMBRANE PROTEINS

Photo system 1 1 Higher plants (vs Rhodohacter sphaeroides) Marchantia psbA Marchantia psbB Marchantia psbC Marchantia psbD Nicotiana psbE Nicotiana psbF Nicotiana psbH Marchantia psbJ Marchantia psbJ Nicotiana psbK Marchantia psbL Cytochrome b61f Marchantia polymorpha (vs Rhodobacter sphaeroides) Cytochrome b6+ Subunit IV (vs R. sphaeroides Cytochrome b) Cytochrome f FeS protein

{

Eukaryote specific

Chlo. Nuc!. Chlo. Nuc!.

Prokaryote specific

Reference

2 5

Subunit H ( l helix)

6 6 5

42 4+ 3 (8)

Photosystem I Algae and higher plants (vs Chlorobium /imico/a)

Marchantia P700 Chlamydomonas P37 ATPase Marchantia polymorph a (vs E. coli) Subunit I Subunit III Subunit IV

91

II

9, 9 1

I

2 5

Antenna H igher plants (vs RllOdobacter

sphaeroides) Pisum LHCII CabI Lycopersicon LHCI Cab I Petunia LHCI CabII Lycopersicoll LHCI CabIII

3 3 3

3

aB870 (I helix) PB870 (I helix) PB80�850 ( l helix) PB80�850 (I helix)

1 54

continued

392

POPOT & DE VITRY

Table 6 (continued)


Complex NADH -Q reductase Marchantia (vs E. coli) Subu nit I Subu nit 2 Subu nit 3 Subu nit 4 Subunit 5 Subu nit 6 Subu nit 4L

Eukaryote & prokaryote

Eukaryote specific

Chlo. Nucl. Chlo. Nucl.

Prokaryote specific

Reference

lSI 7 15

3

14 17

Only one subunit (47 kDa, o helix)

5

3

" Subunits are divided into those common to eukaryotes and prokaryotes, those specific to eukaryotes and th ose specific to prokaryotes (on the basis of presently available sequences). They are further dis

their site of synthesis. The number of helices predicted for each subunit is indicated. The two integral membrane subunits of this protein have similar molecular weights in Bos tauris mitochondria and E. coli but have been sequenced only in E. coli. The number of helices pred icted is indicated for E. coli subunits. Nicotinamide nudeutide Iranshydrogenuse: Thi s protein is composed of one polypeptide in Bos tauris mitochondria and two in E. coli (of Tables I and 4B). Cytochrome b olf: Cytochrome h in the QH2-cytochrome c reductase c omplex of Rhodohacter sphaeroid es corresponds to two subunits [cytochrome ho and subunit IV (braced in table)] in cytochrome hoi/complex (cf Tab les I and 4C). A TPase subunit I: I n contrast to ATPase subunit 8 of mitochondria, subunit I of chloroplast CF 0 shows some simi larity in primary structure to subunit b of E. coli F o. A TPase subunit IV: Regarding the number of putative transmembrane IX-helices, see Footnote d for Table 4. tributed according to

Suainafe-Q reductase:

subunit is synthesized. In mitochondrion inner membrane, there is a clear cut discrepancy: with few exceptions, subunits are imported if they contain three or fewer putative transmembrane !X-helices and are synthesized i n situ if they contain more than three. In chloroplast thylakoids, imported proteins also have few hydrophobic segments, but proteins synthesized i n situ can have either many or few. These distributions d o n o t indirectly result from a tendency for large genes to remain in the organelles, since most soluble or extrinsic proteins, whether large or small, are synthesized in the cytoplasm (3, 93, 1 25). The exclusion is not absolute; there exist at least two natural and one engineered exceptions. These are discussed below. Nevertheless, the tendency of imported proteins to have few trans membrane segments is very strong. The causes of this distribution are uncertain, and may involve several factors. The possibility of mistargeting was proposed by von Heijne ( 1 4 1 a, 1 42), who argued that if a hydrophobic segment appeared in the cytosol before the synthesis of a nuclear-encoded protein is completed, it would



393

act as a signal sequence and target thc protcin to the endoplasmic retic ulum. This could occur if there are more than 70-90 residues after the end of the first hydrophobic segment. M ore recent data, however, suggest that mistargeting cannot be a decisive factor because, at least under this simple form, this hypothesis predicts the misdirection of about half of the imported proteins (marked with a degree sign in Table 4). Other possible explanations might involve the mechanism of import into the mitochondrion or chloroplast. Considerable evidence indicates that import is, primarily or totally, posttranslational (for reviews, see 6, 52, 1 06, 1 4 1 ) . Import involves unfolding of the protein to be translocated. It is prevented by stabilization of the mature, folded conformation ( 1 8, 3 1 , 33, 1 2 1). Cytosolic proteins are involved in preventing folding or aggregation of the nascent chains and/or in unfolding the chains into an import-competent form (e.g. 32, 34, 5 1 , 52, 98, 1 00, 1 1 7). Similar proteins play a role in protein folding and assembly in the mitochondrial matrix ( 1 9, 95, 1 1 2) . The presence of a large number of hydrophobic residues in a polypeptide can perturb import in several ways. To prevent aggregation and pre cipitation or nonspecific association with membranes or with other protcins, large hydrophobic patches must not be exposed to the cytosol. This can be achieved either by appropriate folding of the protein or by association with itself or with other proteins. Particular difficulties are expected for integral membrane proteins because, in contrast to soluble proteins, they expose a considerable hydrophobic area to their surface in their native state. The achievement of a soluble conformation or complex should become increasingly more difficult as the number of hydrophobic segments to be masked increases. Problems also might arise at the unfold ing step, since hydrophobic residue burial is a major sourcc of stabilization free energy for folded structures and for oligomers. The more hydrophobic residues have been buried, the more difficult it is to unfold or dissociate the resulting structure. Similar difficulties may also be encountered on the matrix or stroma side of the organelles. The recent description of a soluble form of the integral protein lac permease ( 1 1 6) or, in contrast the hydrophobic properties of bacterial porins can serve as reminders that the behavior of membrane proteins does not always match our expectations. To surmise that large hydrophobic peptides may stand greater chances of misfolding and precipitating before being targeted to the organelle, or may become untractably difficult to unfold seems reasonable, however. Another conceivable difficulty is the probable tendency of hydrophobic segments located away from the region being translocated to insert nonspecifically into nearby membranes. It is probably significant that imported segments tend to be less hydrophobic


394

POPOT & DE VITRY

than those in subunits synthesized in the organelles (even though the two distributions overlap). This tendency further decreases the total hydro phobicity of the putative transmembrane region in imported proteins. The involvement of hydrophilic regions, e .g. presequences, in stabilizing the precursor form of imported integral proteins has been discussed by Hartl et al (52). Borst ( 1 2) seems to have first proposed the idea that the biosynthesis of some proteins within organelles rather than in the cytosol could be linked to their hydrophobicity. This view has remained in relative disfavor, pre sumably because the interest in the process of translocation itself has focused attention on local properties of the sequence. The example of ATPase subunit 9 shows that local hydrophobicity in imported proteins can be very high (Table 4B). The critical importance of the unfolding step, however, may explain why an accumulation of sequence segments that individually would be importable might have an inhibitory effect. The tentative rule that proteins with more than three hydrophobic transmembrane segments are not imported presently has two natural exceptions. One is nicotinamide nucleotide transhydrogenase, an enzyme from the inner mitochondrial membrane. The other is the phosphate trans locator from the inner envelope membrane of chloroplasts. NNT contains probably 1 2 and the translocator 7 hydrophobic segments (cf Table 4) whose hydrophobicity is typical of that of imported proteins. It might be interesting to determine whether the import of these proteins is posttranslational or coupled to their synthesis. Whatever the reason(s), in most cases organelles do not import proteins with large transmembrane regions. Eukaryotic cells generally have sup plemented complexes inherited from the original symbiotic prokaryotes with additional subunits. In the mitochondrial respiratory chain, all of the new material encoded in the nucleus is made up of I -helix subunits (Table 6). In addition, the genes for some of the smaller, 1 -3-helix integral sub units of prokaryotic origin have been displaced to the nucleus. In chlo roplasts, the imported material is made up of 1-3-helix proteins; most of the new subunits are locally encoded and can have either many or few hydrophobic segments. Many of the hypothetical proteins encoded by ORFs are predicted to be small, I -helix proteins. The existence of many I -helix subunits encoded in plastid DNA indicates that restriction on import is not the only circumstance in which one encounters such subunits. The building up of organelle complexes seems to take full advantage of the domainlike behavior of transmembrane a-helices: they are put together in a piecemeal manner by a process of microassembly that uses numerous small subunits in addition to a few large ones.


395


Other Membranes

Very small I -helix integral subunits are rare among plasma membrane proteins (2 in our sample of 80). Part of the reason for this near absence could be methodologic (such proteins migrate with the dye front in most commonly used SDS-PAGE systems), and part is certainly linked to the different functions of this membrane (see section on results). Differences in biosynthesis may also play a role. It is not clear yet to what extent insertion of proteins into the endoplasmic reticulum is co- or post translational (for recent discussion, see 43). Similarities between insertion into the ER and import into organelles are certainly greater than previously recognized (for reviews, see 1 06, 1 4 1 ). For instance, evidence of a role for stress proteins in yeast has recently been obtained (2 1 , 32, 1 53). However, translation and insertion seem more closely coupled in the ER than for organelle proteins. The red blood cell glucose transporter, a protein thought to contain 1 2 transmembrane segments, can be imported post translationally into dog pancreas microsomes, albeit with a low efficiency. Cotranslational insertion or engineered shortening of the polypeptide by 4 transmembrane segments increases efficiency (85). There are conceivable advantages to using several small subunits instead of a single large one from, for example, evolutionary or regulatory points of view. As mentioned above, numerous small I -helix proteins are syn thesized in situ in chloroplasts. Restriction to import cannot be the reason for their abundance. The scarcity of very small plasma membrane proteins is likely due in part to the absence of the restriction on helix number that seems to be associated with posttranslational import and insertion. It may also be that, in the plasma membrane, any other potential advantage of microassembly is offset by the greater instability of complexes as compared with single-chain proteins or by the increased complexity of targeting and assembly. We have examined under identical conditions the protein composition of another membrane toward which export of hydrophobic proteins is known to present difficulties, namely the outer membrane of gram-negative bacteria (for reviews, see 7, 1 06, 1 07, 1 1 0) . Export or membrane integration of proteins in bacteria presents similarities with import into organelles in that it can be posttranslational ( 1 45); it is prevented by stabilization of the mature, folded form (97, 1 09) and it involves ATP-dependent antifolding proteins (23, 27, 28, 74a). We have analyzed the sequences of 1 1 outer membrane proteins, none of which is thought to form transmembrane hydrophobic a-helices (Table 7). In agreement with the literature, most contained no significantly hydro-

396

POPOT & DE VITRY


Table 7 Hydrophobicity analysis of integral proteins from the outer membrane of E. coli"

Protein

Nr

GES

M urein-lipoprotcin Phospholipase A OmpF (porin) OmpC (porin) PhoE OmpA TolC FhuA (TonA) BtuB LamB (maltoporin) Lc

58 260 340 346 330 325 467 714 594 421 342

- 1 .28 0.60 0.34 0.21 0.42 0.85 1 .27 1 .27 0.95 0.36 0.39

--

---

Reference NBRF NBRF NBRF 83 NBRF NBRF NBRF N B RF 58 NBRF NBRF

---

--

" The number of residues (Nr) and the hydrophobicity of the most hydrophobic 1 7-residue segment (GES; kcaljresidue) are given for the mature protein.

phobic segments at all, and two contained a mildly hydrophobic segment with a GES barely higher than 1 .2. That the bacterial cell has difficulties exporting hydrophobic proteins is directly substantiated by experiments in which stretches of hydrophobic residues were introduced genetically into the sequence of either a viral coat protein, the natural anchoring segment of which had been deleted (30), or an outer membrane protein (80). In both cases, export was blocked as the length of the hydrophobic insert increased. We have estimated the local hydrophobicity of the 1 7residue segments that included these inserts, using the same procedure as for natural proteins. Within some variability, segments with GES values less than 1.6 allowed export and segments with GES values higher than approximately 2.2 blocked it. Partial exportation was observed between these two limits. On this basis, only half a dozen of the 1 40 proteins listed in Table 4 could conceivably be exported efficiently by E. coli. Porins are the best known outer membrane proteins. They have rather polar sequences and are known to be essentially comprised of f3 sheets (for recent review, see 8). A strong restriction on the export of hydrophobic segments may explain why the structural solution adopted by porins differs from the micro assembly observed in organelle complexes. On the other hand, factors such as the peculiar structure, function and environment of bacterial outer membranes should not be forgotten. The sequence of the porin from yeast mitochondrial outer membrane-a protein apparently


397

unrelated to bacterial porins (82a)-is also fairly hydrophilic. The GES value of its most hydrophobic 1 7-residue segment is only 0.72. Displacing the Syn thesis of Integral Membrane Proteins


from Organelles to the Cytoplasm

The observations summarized here may shed light on the conditions under which a protein synthesis can be displaced from organelle to cytoplasm, either during the course of evolution (see 46) or as the result of genetic engineering (cf 36). As already mentioned, proteins like NNT or the chloroplast envelope phosphate translocator appeared atypical in our analysis. Perhaps their biosynthesis presents peculiarities-for instance a closer coupling between translation and import. One can also wonder whether differences exist between homologous proteins depending on their site of synthesis. For example, some complex I subunits synthesized in situ in mammalian mito chondria are presumably imported from the cytoplasm in the parasitic protozoa Leishmania and Trypanosoma (Table 5). Does the average hydro phobicity of the transmembrane segments in imported segments diminish? Are proteins with many transmembrane segments split into several smaller ones? Only a few cases of displaced subunits can presently be analyzed from this point of view. Within eukaryotes, comparison of a subunit imported from the cytosol with an equivalent one synthesized in situ is possible for ATPase subunit 9 and for cytochrome c , . The average hydrophobicity of the two transmembrane segments of ATPase subunit 9 is similar whether the protein is encoded in the nucleus (as in mammals and Neurospora), in the mitochondrion (as in yeast and maize), or in the chloroplast. In contrast, the hydrophobicity of the putative transmembrane helix of mito chondrial cytochrome c " which is encoded in the nucleus, is much lower than the hydrophobicity of the equivalent segment in cytochromcf, which is encoded in the chloroplast (respective GES values 1 . 53 and 2.26). Comparison of eukaryotic and prokaryotic complexes shows that the following integral subunits have been displaced to the eukaryote nucleus (Table 6): cytochrome c , and FeS subunits of the QH2-cytochrome c reductase complex, ATPase subunit 9, and NNT. Again, the hydro phobicity of the anchoring sequence of cytochrome c , is found to be much higher when it is not imported (GES 2.24 in E. coli vs 1 . 53 in eukaryotes). In the other cases, the hydrophobicity remains about the same regardless of import. It is probably premature to draw conclusions from such a limited comparison, particularly as it does not include 3-helix proteins, which presumably would be most sensitive to selective pressure on their hydrophobicity. =


398

POPOT & DE VITRY

We have as yet no examples of a protein with many transmembrane segments that would be split into several smaller ones when its structural gene is displaced to the nucleus. We are aware of only two natural cases of split integral proteins (Table I), if one leaves aside voltage-gated chan nels from the plasma membrane, in which a homooligomer in one case (K + channel) appears to correspond to a single polypeptide with internal repeats in others (Na + and Ca 2+ channels; cf Table 4A). In the case of cytochrome b, neither the whole polypeptide nor the fragments need be imported. In case of NNT, the fragments are not imported while the fUll length protein is. The existence of restrictions to integral protein import can be exper imentally tested by displacing the locus of synthesis of organelle-encoded proteins to the cytoplasm. Nagley et al (87) found nucleus-encoded subunit 8 of the Fo ATPase (fused to a mitochondrial targeting peptide) to rescue yeast mutants lacking functional mitochondrial subunit 8. This obser vation does not test the ideas developed here, as subunit 8 is a very short protein (48 amino acid residues in yeast) purported to comprise a single transmembrane segment ( 1 38). Its structural gene is absent from Xenopus mitochondrial genome ( 1 1 5), which suggests that in this organism it is naturally encoded in the nucleus. Such is not the case for D l , the photosystem II quinone-binding protein that carnes the site of action of the herbicide atrazine. D l is encoded by the psbA gene, which is present in every chloroplast genome sequenced thus far (93, 1 25). This protein most likely features five transmembrane seg ments (82, 1 37). Cheung et al (20) have reported that introduction of the psbA gene from an atrazine-resistant biotype into the tobacco nuclear genome conferred an increased tolerance to atrazine to some of the trans formed plants. This observation suggests that the existence of an absolute barrier to the import of D l is not the reason for retention of the psbA gene in the chloroplast. The efficiency of the import was not established directly and was difficult to assess from functional data because the engineered protein had to compete with the natural one whose synthesis was not blocked. Such a competition could explain the limited resistance to atrazine of the engineered strains. Further experiments are needed to establish to what extent efficient import can be achieved for mUltispanning proteins. Our data suggest that low yields of import may be encountered. One attractive possibility for molecular genetic experiments involves splitting genes coding for polytopic proteins into two or more smaller parts, each preceded by a segment coding for an organelle targeting peptide. It is not unreasonable to expect that the resulting protein fragments could assemble in the organelle inner membrane into functional complexes.

399



CONCLUSION

The present analysis supports the idea that transmembrane a-helices rep resent autonomous foiding domains in integral membrane proteins. It further suggests the existence of biosynthetic problems associated with the posttranslational import or export of proteins containing long stretches of hydrophobic residues. In organelles, restrictions to import are not absolute, and these problems are circumvented by importing numerous small subunits containing few hydrophobic segments. These are sub sequently microassembled into complexes thanks to the domainlike behavior of transmembrane a-helices. In the endoplasmic reticulum, microassembly is presumably not required and is in fact seldom observed. ACKNOWLEDGMENTS

We are grateful to D. M . Engelman, J.-P. Henry, P. Joliot, M. Le Maire, W. Neupert, F. Pattus, M. Uzan and G. von Heijne for comments on the manuscript and discussions, to the late P. Klein for a copy of the source code of the program described in Ref. 70 and to D. Beal for his help with the graphic system used to generate the figures. Sequence retrieval and homology analyses were performed using computer facilities at the CITI2 with the help of the Ministere de la Recherche et de la Technologie. This work was supported by a grant from the Ministere de la Recherche et de la Technologie to J.-L. Popot.

Literature Cited I . Addison. R. 1 986. 1. Bioi. Chern. 261 :

2. 3.

4. 5. 6. 7. 8. 9.

14896-14901 Allen, J. P., Feher, G., Yeates, T. 0., Komiya, H., Rees, D. C. 1 987. Proc. Natl. Acad. Sci. USA 84: 6 1 62-

Fragmentation of integral membrane proteins in the gas phase.

Thermodynamics, the structure of integral membrane proteins, and transport.

Lateral mobility of human erythrocyte integral membrane proteins.

Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

Comparative genomic analysis of integral membrane transport proteins in ciliates.

Neurotransmitter transporters. A novel family of integral plasma membrane proteins.

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins.

Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Comprehensive Phylogenetic Analysis Sheds Light on the Diversity and Origin of the MLO Family of Integral Membrane Proteins.

Preparation of tethered-lipid bilayers on gold surfaces for the incorporation of integral membrane proteins synthesized by cell-free expression.

Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents.

Click Chemistry Reagent for Identification of Sites of Covalent Ligand Incorporation in Integral Membrane Proteins.

Role of Lipids in Folding, Misfolding and Function of Integral Membrane Proteins.

Mechanism for transforming cytosolic SOD1 into integral membrane proteins of organelles by ALS-causing mutations.

Perfusion chromatography-a new procedure for very rapid isolation of integral photosynthetic membrane proteins.

High-throughput cloning and expression of integral membrane proteins in Escherichia coli.

Monte Carlo simulation studies of lipid order parameter profiles near integral membrane proteins.

Sorting of integral membrane proteins mediated by curvature-dependent protein-lipid bilayer interaction.

Lateral and vertical displacement of integral membrane proteins during lipid phase transition in Anacystis nidulans.

High-performance hydroxyapatite chromatography of integral membrane proteins and water-soluble proteins in complex with sodium dodecyl sulphate.

A time integral of membrane currents.

Integral membrane proteins and free electron lasers - a compatible couple indeed!

The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

Lipid-like Peptides can Stabilize Integral Membrane Proteins for Biophysical and Structural Studies.