The proteome of rat olfactory sensory cilia

322 DOI 10.1002/pmic.200800149 

Proteomics 2009, 9, 322–334 

RESEARCH ARTICLE 

The proteome of rat olfactory sensory cilia 

Ulrich Mayer 1 , Alexander Küller 2 *, Philipp C. Daiber 1 , Inge Neudorf 1 , Uwe Warnken 3 , 

Martina Schnölzer 3 , Stephan Frings 1 and Frank Möhrlen 1 

1 Department of Molecular Physiology, Institute of Zoology, University of Heidelberg, Heidelberg, Germany 

2 Department of Applied Physical Chemistry, Institute of Physical Chemistry, University of Heidelberg, 

Heidelberg, Germany 

3 Functional Proteome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany 

Olfactory sensory neurons expose to the inhaled air chemosensory cilia which bind odorants and 

operate as transduction organelles. Odorant receptors in the ciliary membrane activate a transduction 

cascade which uses cAMP and Ca 21 for sensory signaling in the ciliary lumen. Although 

the canonical transduction pathway is well established, molecular components for more complex 

aspects of sensory transduction, like adaptation, regulation, and termination of the receptor response 

have not been systematically identified. Moreover, open questions in olfactory physiology 

include how the cilia exchange solutes with the surrounding mucus, assemble their highly 

polarized set of proteins, and cope with noxious substances in the ambient air. A specific ciliary 

proteome would promote research efforts in all of these fields. We have improved a method to 

detach cilia from rat olfactory sensory neurons and have isolated a preparation specifically enriched 

in ciliary membrane proteins. Using LC-ESI-MS/MS analysis, we identified 377 proteins 

which constitute the olfactory cilia proteome. These proteins represent a comprehensive data set 

for olfactory research since more than 80% can be attributed to the characteristic functions of 

olfactory sensory neurons and their cilia: signal processing, protein targeting, neurogenesis, 

solute transport, and cytoprotection. Organellar proteomics thus yielded decisive information 

about the diverse physiological functions of a sensory organelle. 

Received: 14 February, 2008 

Revised: 16 July, 2008 

Accepted: 1 August, 2008 

Keywords: 

Mass spectrometry / Olfactory receptor neurons / Proteomic analysis / Sensory cilia / 

Signal transduction 

1 Introduction 

Correspondence: Dr. Frank Möhrlen, Department of Molecular 

Physiology, University of Heidelberg, Im Neuenheimer Feld 230, 

69120 Heidelberg, Germany 

E-mail: moehrlen@uni-hd.de 

Fax: 149-6221-54-5627 

Abbreviations: AC III, adenylyl cylase type III; CNG, cyclic nucleotide 

gated; FA, formic acid; OR, odorant receptor; ORN, olfactory 

receptor neuron; SEM, scanning electron microscopy 

In the mammalian nose, odorants are detected by primary 

afferent olfactory receptor neurons (ORNs). These cells 

project a single dendrite to the surface of the olfactory neuroepithelium 

where a tuft of sensory cilia protrudes from 

the dendritic ending into a mucus layer, the interface between 

air and tissue. Odorants dissolve in this mucus and 

make contact with the ciliary membrane where they bind to 

receptor proteins and trigger a metabotropic transduction 

cascade. The canonical transduction pathway (reviews: [1, 

2]) begins with adenylyl cylase type III (AC III) which is 

activated by odorant receptors (ORs) via a stimulatory GTPbinding 

protein (G olf ) and synthesizes cAMP as second 

messenger. cAMP opens Ca 21 -permeable transduction 

channels which assemble in the ciliary membrane from the 

* Current address: CeloNova BioSciences Germany GmbH, Im 

Neuenheimer Feld 515, 69120 Heidelberg, Germany. 

© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2009, 9, 322–334 323 

three subunits CNGA2, CNGA4, and CNGB1b. The Ca 21 

influx through these cyclic nucleotide-gated (CNG) channels 

leads to opening of ciliary Ca 21 -activated chloride 

channels which amplify the receptor potential and cause 

electrical excitation. While major components of this transduction 

chain are known, there are many gaps in our 

knowledge which hinder progress in olfactory research 

considerably. For example, the main charge carrier of the 

olfactory receptor current is chloride, but the molecular 

identity of the relevant chloride channel has not yet been 

established with certainty [3–5]. The homeostasis of chloride 

is of central importance for ORN function, but it is not clear 

which chloride transporters are responsible [6–9]. Various 

molecular candidates have been suggested to contribute to 

ORN adaptation, but a definite role was not proven for any 

of them [10–14]. In addition to these sensory tasks, a continuous 

process of adult neurogenesis upholds the total 

number of ORNs, which live for only a few weeks, and 

maintains the ordered connectivity to the olfactory bulb of 

the brain [15, 16]. Moreover, the assembly of the cilia and 

the targeting of ciliary membrane proteins is an emerging 

topic in olfactory research [17, 18]. And finally, it has 

become clear in recent years that several distinct types of 

receptor neurons coexist in the olfactory epithelium, each 

serving a specific sensory purpose and operating with a 

specific set of signal processing proteins [19–21]. In view of 

the increasingly complex protein networks that constitute 

our concepts of olfaction, a survey of protein expression in 

ORN cilia would be helpful as reference for future studies. 

Such a survey can be obtained from a proteomic analysis of 

isolated cilia of sufficient purity. 

In a recent study, we have analyzed the purity of the 

cilia preparation routinely used in olfactory biochemistry 

[22]. For this preparation, ORN cilia are separated from 

olfactory epithelium by a calcium shock and harvested by 

ultracentrifugation. We characterized 268 proteins in this 

preparation and allocated these proteins to the different 

cell types and subcellular compartments of the olfactory 

epithelium. The concentration of ciliary marker proteins 

revealed that the calcium-shock preparation was highly 

enriched in ciliary membranes. However, almost half of the 

protein set did not originate from ciliary membranes but 

represented contaminations from nonciliary structures. 

Therefore, the conventional cilia preparation is not the appropriate 

material for the ciliary protein survey intended 

here. 

In the present study, we improved the method of cilia 

isolation and documented the quality of deciliation by scanning 

electron microscopy (SEM) and immunohistochemistry. 

An advanced LC protocol prior to MS was then used to 

characterize the cilia proteome of rat ORNs. The result is a 

comprehensive data set of 377 proteins most likely originating 

from olfactory cilia. Functionally, these proteins could be 

attributed to olfactory signal processing, neurogenesis, ciliary 

protein targeting, transport processes, and cytoprotection. 

2 Materials and methods 

2.1 Animals 

Preparations of rat olfactory epithelium and olfactory cilia 

were obtained from 3 to 6-month-old Wistar rats. Animals 

were killed by CO 2 inhalation and decapitation. All experiments 

were performed in accordance with the Animal Protection 

Law and the guidelines and permissions of the University 

of Heidelberg. For immunohistochemical treatment, 

the rostral part of the skull, containing the nasal cavity, was 

dissected. For membrane preparations, the head capsule was 

opened by a sagittal section to remove the olfactory epithelium 

from the dorsal posterior part of the nasal septum. 

2.2 Immunohistochemistry and microscopy 

Immunohistochemistry of cryosections from rat olfactory 

epithelium was performed as previously described [23]. The 

following primary antibodies and dilutions were used: rabbit 

anti-AC III 1:500 (Santa Cruz Biotech, #sc-588), goat-anti 

ezrin 1:50 (Santa Cruz Biotech, #sc-6409), mouse monoclonal 

anti-SLC4A1 1:50 (Abcam, BIII 136, #ab11012), rabbit 

anti-Flotilin-1 1:250 (Santa Cruz Biotech, H-104, #sc-25506), 

rabbit antiplexin B2 1:50 (Santa Cruz Biotech, H-90, #sc- 

67034), and rabbit anti-CLIC6 1:500 (AVIVA Systems Biology, 

#ARP35388). Visualization of primary antibodies was 

performed using donkey antirabbit Alexa488 (Molecular 

Probes, A-21206, 1:500), donkey antigoat Alexa568 (Molecular 

Probes, A-11057, 1:500), and goat antimouse Alexa 568 

(Molecular Probes, A-11004, 1:500) secondary antibodies. A 

1:300 dilution of a 90 mM DAPI solution (Molecular Probes, 

C-7509) was used to stain nuclei. Appropriate controls were 

performed for all antibodies used. Sections were analyzed 

using a Nikon Eclipse E600 FN epifluorescence microscope 

with a PCO SensiCam 12 Bit Cooled Imaging digital camera. 

2.3 SEM 

Isolated olfactory tissue was washed for 5 min in solution A 

(140 mM NaCl, 3 mM KCl, 2 mM MgSO 4 , 7.5 mM D-glucose, 

20 mM HEPES, pH 7.4) or B (140 mM NaCl, 2 mM MgSO 4 , 

7.5 mM D-glucose, 20 mM HEPES, pH 7.4) each containing 

5 mM EGTA. After treatment with solutions A, C (20 mM 

CaCl 2 in A), or D (20 mM CaCl 2 , 30 mM KCl in B), the tissue 

was placed in fixative for 2 h (2.5% w/v glutaraldehyde in 

0.1 M sodium cacodylate buffer, pH 7.4). Tissue was postfixed 

for 2 h in 1% v/v osmium tetroxide/1.5% v/v potassium 

hexacyanoferrat. Following fixation and washing in 0.05 M 

maleate buffer, pH 2.5, the specimen was dehydrated in a 

graded series of aceton, critical point dried (BAL-TEC CPD 

030, critical point dryer) with several wash-and-rinse cycles of 

CO 2 , sputter-coated with gold (BAL-TEC, MED 020 coating 

system, 15 nm, 5610 22 mbar argon), and examined in a 

Zeiss LEO 1530 scanning electron microscope at an accelerating 

voltage of 3 kVand a base pressure of ,5610 26 mbar. 


324 U. Mayer et al. Proteomics 2009, 9, 322–334 

2.4 Isolation of olfactory cilia 

Isolation of olfactory cilia was performed both using (i) the 

established “calcium-shock” method as described in ref. 

[22] and (ii) a new protocol combining a Ca 21 /K 1 -shock 

with NaBr treatment, subsequently designated as “Ca 21 / 

K 1 -shock” method. The latter protocol starts with washing 

the olfactory epithelia of seven rats in ice-cold solution B 

containing 5 mM EGTA and 1% protease-inhibitor-mix 

(50 mM PMSF, 10 mM iodoacetamide, 100 mM pepstatin 

A, 10 mM o-phenanthroline, solubilized in DMSO). Afterwards, 

olfactory cilia were detached by gentle agitation in 

deciliation solution (solution B containing 20 mM CaCl 2 , 

30 mM KCl, and 1% protease-inhibitor-mix) for 20 min at 

47C. Sequential centrifugation at 8506g and 65006g for 

10 min at 47C was performed to remove pieces of tissue. 

The supernatant containing detached cilia was collected 

and loaded on top of a 45% w/v sucrose solution in deciliation 

solution, and was concentrated at 100 0006g for 

60 min at 47C using a Beckman L-70 ultracentrifuge. Cilia 

were visible as a pallid white layer at the sucrose–supernatant 

interface, and were diluted in ten-fold volume of 

deciliation solution and centrifuged at 100 0006g for 

60 min at 47C. The resulting cilia pellet was sequentially 

resuspended in 2 and 1 M NaBr solution and each centrifuged 

at 100 0006g for 60 min at 47C. Finally, the cilia 

were resuspended in washing solution (10 mM Tris, 3 mM 

MgCl 2 , 2 mM EGTA, pH 7.4) and centrifuged at 

100 0006g for 60 min at 47C. The protein yield was measured 

using the Bradford assay [24] and resulted in 10 mg 

protein per seven rats. 

2.5 Whole tissue membrane protein purification 

Whole membrane protein extracts of the olfactory epithelium 

were prepared as described in ref. [22]. 

2.6 Immunoblot 

Six micrograms of proteins from cilia detached by calciumshock 

or Ca 21 /K 1 -shock and proteins from whole membrane 

extracts were loaded on 10% 1-D SDS gels [25], electroblotted 

to PVDF membranes (Machery & Nagel; Germany) and 

incubated with appropriate antibodies as described in ref. 

[22]. The following antibodies and dilutions were used: rabbit 

anti-AC III 1:200 (Santa Cruz Biotech, #sc-588); mouse anti- 

CNGA4 1:20 (mAB7B11, monoclonal, directed against c-terminal 

residues 392–575; developed by [12]; obtained from 

the Developmental Studies Hybridoma Bank developed 

under the auspices of the NICHD and maintained by The 

University of Iowa, Department of Biological Sciences, Iowa 

City, IA 52242); goat antiezrin 1:200 (Santa Cruz Biotech, 

#sc-6409). The ECL plus enhanced chemoluminescence system 

(Amersham Biosciences, UK) was used to monitor 

bound antibodies. 

2.7 MS 

Proteins of cilia derived from the Ca 21 /K 1 -shock method 

were separated on a denaturing 7–12.5% gradient SDS gel 

according to [25] and stained with colloidal Coomassie CBB 

G250 using Roti © -Blue (Roth, Karlsruhe, Germany). The 

resulting gel was cut into 29 pieces and digested with trypsin 

(Promega, Madison, WI). The supernatant from the tryptic 

digestion and the extracts from step 1 (acetonitrile (MeCN)/ 

H 2 O/formic acid (FA), 50.0/49.9/0.1 v/v/v), 2 (MeCN 100%), 

3(H 2 O/FA, 99.9/0.1 v/v), and 4 (MeCN/H 2 O/FA, 50.0/49.9/ 

0.1 v/v/v) were combined, evaporated, and dissolved in H 2 O/ 

FA, 99.9/0.1 v/v. 

Nanoscale LC-MS analysis of the extract aliquots was 

performed using the CapLC capillary LC system (Waters, 

Eschborn, Germany) which was coupled to a hybrid quadrupole 

orthogonal acceleration TOF tandem mass spectrometer 

(Q-TOF; Micromass, Manchester, UK). The LC-ESI-MS/ 

MS device was adjusted with a PicoTip Emitter (New 

Objective, Woburn, MA, USA) fitted on a Z-spray (Micromass) 

interface. For peptide trapping, a Symmetry 300 

NanoEase C18 column (Waters) was used. Chromatographic 

separations were performed on an RP capillary column 

(Atlantis C18, 3 mm particle size, 75 mm inner diameter, 

15 cm length (Waters) with a flow rate of 200 nL/min. 

For all gel slices (Fig. 2B), the chromatography was carried 

out using a long linear gradient from solvent A (99.9% 

v/v H 2 O, 0.1% v/v FA) to 55% solvent B (5% v/v H 2 O, 94.9% 

v/v MeCN, 0.1% v/v FA) in 2.3 h and from 55 to 100% solvent 

B in 3.8 h. A nanoelectrospray ion source was used for 

ionization of eluted peptides. The cone voltage was set to 

80 V and the capillary voltage was set to 2400 V. Data acquisition 

was controlled by MassLynx 4.0 software (Waters). 

Low-energy CID was performed with argon as a collision gas 

(pressure in the collision cell was set to 5610 25 mbar), and 

the collision energy was optimized for all precursor ions dependent 

on their charge state and molecular weight in the 

range of 25–40 eV. Mass Lynx raw data files were processed 

with Protein Lynx Global Server 2.2 software (Waters). Deisotoping 

was performed using the MaxEnt3 algorithm. 

2.8 Protein Identification 

Processed data were searched against the rat protein subdatabase 

(37 521 protein sequences) of the National Center 

for Biotechnology Information (NCBI) nonredundant 

(120 506; 4 196 452 protein sequences) database using the 

MASCOT algorithm version v2.1 (Matrix Science, London, 

UK). The mass tolerance was set to 0.1 Da for fragment ions 

and 200 ppm for precursor ions. No fragment ions score cutoff 

was applied. The following search parameters were 

selected: variable modification due to methionine oxidation, 

fixed cysteine modification with the carbamidomethyl-side 

chain, one missed cleavage site in the case of incomplete 

trypsin hydrolysis. MASCOT result files were analyzed using 

the software MASCOT file analyzer (MFA), available at 


Proteomics 2009, 9, 322–334 325 

http://www.zoo.uni-heidelberg.de/mfa/PIC. The following 

settings were applied: annotation threshold60%, minimum 

sum score 50, minimum number of peptides 2. 

Furthermore, protein hits were taken as identified if a minimum 

of one peptide had an individual ion score exceeding 

the MASCOT identity threshold. Under the applied search 

parameters a sum MASCOT score of 50 typically refers to a 

match probability of p0.0005, where p is the probability that 

the observed match is a random event. Redundancy of proteins 

that appeared in the database under different names 

and accession numbers was eliminated. 

2.9 Bioinformatics 

For fast information procurement on each annotated protein, 

we used Protein Information Crawler (PIC; [26]). For 

further annotation of protein sequences, Basic Local Alignment 

Search Tool (BLAST) searches [27, 28] at NCBI and 

sequence based Harvester searches [29] were used. Prediction 

of transmembrane helices was performed by using 

TMHMM Server v. 2.0 [30] and SOSUI [31]. For subcellular 

localization, the Subcellular Localization Database Locate 

was used [32]. All analyses were complemented by evaluating 

peer-reviewed publications. 

3 Results 

3.1 Preparation of ciliary proteins 

Our rationale for improving the purity of the olfactory cilia 

preparation was to achieve selective isolation of cilia while 

leaving the epithelial tissue unaffected. We monitored the 

efficiency of the deciliation protocols using SEM. Figure 1A 

shows that the surface of untreated tissue is covered with the 

tubular cilia structures as well as with numerous small glo- 

Figure 1. Quality of deciliation by calcium shock and Ca 21 /K 1 -shock. (A) SEM image of control olfactory neuroepithelium with sensory cilia 

(tubular structures) and mucus globules (surface view). The inset gives a closer look on the ciliary structures. Both scale bars indicate 2 mm. 

(B) Coronal cryosection showing the polarized expression of AC III (green), used as a ciliary marker protein in this study. (C) Colocalization 

of AC III with the microvilli marker protein ezrin (red) at the apical rim of the epithelium. (D) The conventional “calcium-shock” deciliation 

method reduces the cilia density, but the remaining mucociliary matrix still covers the surface (see inset). (E) The AC III signal is partly 

removed but much of the ciliary matrix remains in place. (F) Both AC III and ezrin signals are reduced, illustrating that the calcium shock 

detaches both cilia and microvilli. (G) Treatment with the Ca 21 /K 1 -shock leaves the apical surface practically free of cilia. Dendritic endings 

can be seen (inset) with stumps of broken-off cilia. (H) The punctate pattern of the AC III immunosignal originates from the remaining 

dendritic endings. (I) While deciliation has virtually abolished the AC III signal, the remaining ezrin signal documents the differential effect 

of the Ca 21 /K 1 -shock on cilia and microvilli. Blue structures in B, E, H are DAPI-stained nuclei. Scale bars indicate 2 mm in the SEM images 

(A, D, G; also insets) and 25 mm in the fluorescence images. 



bules scattered across the apical surface. These globules 

result from the fixation of the olfactory mucus, as a wash 

with the mucolytic agent DTT (3 mM) partly removed these 

structures (not shown). The apical endings of the ORN dendrites 

from which the cilia emanate are not visible in the 

dense ciliary matrix (inset to Fig. 1A). This matrix gives an 

intense immunosignal for AC III in coronal cryosections of 

the olfactory epithelium (Fig. 1B), reflecting the polarized 

expression of AC III in the ciliary membrane. Examining the 

tissue after the calcium-shock treatment revealed that the 

ciliary matrix was only partially removed. The apical surface 

was still densely ciliated (Fig. 1D), and dendritic endings 

were mostly concealed underneath the cilia (inset to Fig. 1D). 

Accordingly, cryosections from treated tissue show an almost 

continuous AC III immunosignal which was, however, 

markedly reduced when compared to the control tissue 

(Fig. 1E). This result shows that much of the ciliary material 

is not detached by the calcium-shock method. 

To increase the yield of ciliary material, we adapted a 

deciliation procedure that was originally developed for the 

detergent-free isolation of cilia from Paramecium [33]. Basically, 

this method consists of keeping the cells in a low-salt, 

Ca 21 -free buffer, before adding CaCl 2 and KCl (10 and 

30 mM, respectively) to trigger the deciliation process. 

Importantly, this procedure did not induce cell lysis or blistering 

in Paramecium, both of which would introduce nonciliary 

proteins into the preparation. With olfactory epithelium, 

we found a combined Ca 21 /K 1 -shock with 20 mM CaCl 2 

and 30 mM KCl most effective for deciliation (see Section 2). 

Tissue treated with this protocol showed a smooth apical 

surface, almost completely devoid of cilia, as well as good 

tissue integrity (Fig. 1G). Many dendritic endings were now 

visible at the apical surface, bearing the stumps of cilia which 

usually broke off near the ciliary base at the dendritic knobs 

(inset to Fig. 1G). In the immunohistochemical analysis, this 

tissue showed a residual, punctate staining pattern at the 

apical rim, consistent with a signal originating from the 

deciliated dendritic knobs. AC III is expressed both in dendritic 

knobs and proximal cilia segments, although at lower 

density than in the distal parts of the cilia [34]. Thus, the 

Ca 21 /K 1 -shock produces a better gain of ciliary material than 

the conventional calcium-shock method. 

A higher gain of ciliary material can result in an 

increased purity of the cilia preparation, provided that the 

Ca 21 /K 1 -shock protocol does not also lead to an increase of 

nonciliary material. To monitor the separation of cilia and 

nonciliary material, we used ezrin, a protein which is located 

in immediate proximity to the cilia (Fig. 1C). Ezrin is not 

expressed in cilia but in the microvilli of the epithelial supporting 

cells. These cells form a single layer at the top of the 

olfactory epithelium where their microvilli are interspersed 

with the ORN cilia. As such, ezrin is a suitable marker protein 

for nonciliary apical membranes. Immunosignals of AC 

III and ezrin completely match in untreated tissue (Fig. 1C), 

as expected from earlier studies [35, 36]. Following the Ca 21 / 

K 1 -shock, the AC III signal is reduced to a punctate pattern 

while the ezrin signal remains strong (Fig. 1I). Thus, the 

Ca 21 /K 1 -shock exerts a differential effect on cilia and microvilli. 

It detaches cilia while leaving the microvilli largely 

intact. In contrast, tissue treated by the conventional calcium-shock 

treatment (Fig. 1F) appears more damaged with 

sections of the microvilli layer missing. These data show that 

the Ca 21 /K 1 -shock method is better suited to separate ciliary 

from nonciliary material. 

To assess the purity of the material, we determined the 

degree of enrichment of two ciliary-marker proteins, AC III 

and CNGA4, in the cilia preparation. Membrane proteins 

were isolated from (i) total olfactory epithelium, (ii) the cilia 

preparation obtained by calcium-shock, and (iii) the cilia 

preparation obtained by the Ca 21 /K 1 -shock. The same 

amount of total protein from each of these preparations 

(6 mg; verified by densitometric analysis of gel stainings) was 

separated by SDS-PAGE, and the amounts of AC III and 

CNGA4 proteins were compared on Western blots. Figure 2A 

shows a representative result for 12 experiments. While the 

signals for the two proteins are hardly detectable in lane I 

(olfactory epithelium with cilia), the much stronger signals 

in lane III (cilia preparation resulting from Ca 21 /K 1 -shock) 

demonstrate a roughly 100-fold enrichment of the marker 

proteins in that preparation. The calcium-shock protocol 

produces less enrichment, as illustrated by the weaker bands 

in Figure 2A (lane II). This result shows that the Ca 21 /K 1 - 

shock method yields an enhanced efficacy of cilia purification. 

Inspection of the ezrin immunosignals (Fig. 2A, bottom) 

showed that 6 mg total protein of cilia preparation contained 

equal (calcium shock; lane II) or less (Ca 21 /K 1 -shock; 

lane III) ezrin than 6 mg of total protein from the complete 

tissue (lane I). This result provides important information 

about the purity of the cilia preparation. While ciliary material 

is roughly 100-fold enriched in the cilia preparation, the 

content of microvillar material is diminished by the Ca 21 / 

K 1 -shock preparation, indicating that the preparation is 

contaminated by ,1% with microvilli. These results 

demonstrate that the cilia preparation obtained by the Ca 21 / 

K 1 -shock protocol is indeed highly enriched in cilia and 

contains only little contaminations from nonciliary material. 

3.2 Characterization of proteins in the cilia 

preparation 

In analyzing the protein spectrum of the cilia preparation, 

our goal was to specifically identify those proteins which are 

associated with the ciliary membrane. Integral membrane 

proteins (like AC III; [37]) and proteins associated with the 

ciliary membrane (like phosphodiesterase PDE1C2; [38]) 

constitute the specific proteome of olfactory function. We 

tried to remove from the preparation soluble proteins which 

can freely diffuse between the ciliary and dendritic compartment 

in vivo, as well as such soluble proteins of nonciliary 

origin that contaminated the preparation during the isolation 

procedure. Ciliary proteins (20 mg) were separated by 

SDS-PAGE using a 7–12% density gradient to cover a large 


Proteomics 2009, 9, 322–334 327 

Figure 2. Characterization of the cilia preparation obtained by the 

Ca 21 /K 1 -method. (A) Analysis of marker-protein contents in the 

cilia preparation by Western blots. The marker-protein content 

was compared between 6 mg total protein of whole olfactory epithelium, 

including cilia (lane I), and 6 mg total protein in cilia preparations 

obtained by either calcium shock (lane II) or by Ca 21 /K 1 - 

shock (lane III). Comparison of lanes I and III reveals a roughly 

100-fold enrichment of the ciliary proteins AC III and CNGA4, and 

a partial depletion of the microvillar marker ezrin, in the cilia 

preparation. In contrast, the conventional calcium-shock method 

yielded less enrichment of ciliary proteins and no depletion of 

ezrin (compare lanes I and II). (B) Separation of ciliary proteins by 

1-D SDS-PAGE (7–12.5% density). Twenty micrograms of protein 

isolated by Ca 21 /K 1 -shock was separated and stained by colloidal 

Coomassie. For MS, the gel was cut into 29 fragments according 

to the indicated pattern. The protein numbers in Supporting 

Information Table S1 correspond to the numbering of the gel 

fragments. 

range of protein sizes. The resulting gel (Fig. 2B) was cut into 

29 sections (3612 mm 2 each) which were analyzed separately 

by LC-ESI-MS/MS. To examine the reliability of our 

protocol, we analyzed the protein content of three representative 

gel pieces (#16, 19, 22) from four different preparations 

by LC-ESI-MS/MS. The gel pieces contained the 

same protein mix in all four gels. The protocol thus yielded a 

reproducible protein distribution on the SDS-PAGE. 

In total, we identified 463 different proteins in this gel. 

The list of all 463 proteins, together with the mass spectrometric 

details and bioinformatic analyses, is supplied as 

Supporting Information (Table S1) and can be downloaded 

from the authors website at http://www.zoo.uniheidelberg.de/prot/UM1148 

or the proteomics website at 

www.proteomics-journal.com. Protein sizes ranged from 11 

to 569 kDa with isoelectric points (pI) between 4 and 12 

(Figs. 3A and B). A plot of protein size against pI (Fig. 3C) 

showed that a wide range of molecular weights was covered 

both by acidic and basic proteins. Figure 3D shows the distribution 

of predicted transmembrane helices in the integral 

membrane proteins. For 45% of these proteins, more than 

one transmembrane helix is predicted by TMHMM and 

SOSUI. Our comprehensive bioinformatic annotation of all 

protein sequences (see Supporting Information Table S1) 

revealed that 43% of the proteins were integral membrane 

proteins, 30% were membrane-associated proteins, and the 

remaining 27% were soluble proteins (Fig. 4A). To compare 

the proteomic results obtained from the Ca 21 /K 1 -method to 

that obtained by the conventional calcium-shock, we set our 

protein list against the one published by [22]. From the 268 

proteins identified in that study, 155 were also detected in 

the present protein list. An analysis of the proteins which 

are absent from our present list of 463 proteins revealed 

that most of the missing 113 proteins were either soluble 

proteins (72) or proteins assigned to different intracellular 

membranes (38). Only 3 of the 113 proteins were located to 

the plasma membrane. In accordance with the enrichment 

of the marker proteins AC III and CNGA4, this comparison 

shows that the deciliation procedure by Ca 21 /K 1 -shock 

leads to a material in which ciliary membrane proteins are 

more efficiently enriched. For further assessment of the 

quality of the preparation, a comparison with the recent 

ORN transcriptome analysis [39] yields a greater than 83% 

match, confirming that the proteins identified are predominantly 

expressed in ORNs. 

Prior to additional analyses, obvious contaminants such 

as serum (6 proteins, 1%) or nuclear (12, 3%) proteins, were 

excluded from the protein list. Small amounts of mitochondrial 

(15, 3%), peroxisomal (8, 2%), endosomal (5, 1%), or 

ribosomal (30, 6%) proteins, which are not part of the ciliary 

compartment, were also removed from the olfactory cilia 

proteome. In sum, these proteins accounted for approximately 

20% of the proteins identified; this is comparable to 

the levels detected in virtually all other cilia and flagellar 

proteomic analyses (see [40, 41]). The remaining 377 proteins 

are listed in Supporting Information Table S2 (for 

download location, see previous paragraph) and represent a 

protein set most likely involved in olfactory function. Eighty 

percent of these proteins are integral membrane proteins, 

membrane-associated proteins, or components of supramolecular 

complexes linked to membranes. Fourteen percent of 

them are cytoskeleton proteins. The presence of 20% soluble 

proteins may be interpreted as a consequence of vesicle formation 

during the preparation (see [42]), which can trap 

cytosolic constituents. Some of these proteins may also 

represent new components of membrane-associated, supramolecular 

complexes which so far evaded identification in 



Figure 3. Properties of proteins identified in the cilia preparation. (A) Molecular weights of the 463 proteins contained in the cilia preparation 

which are distributed over a total range from 11 to 569 kDa. (B) The distribution of pI covers a range from 4 to 12 with peaks for slightly 

acidic and basic proteins. (C) Plotting molecular weight against pI illustrates that the entire pI range is represented by proteins ,100 kDa, 

while larger proteins tend to have acidic pIs. (D) Distribution of the numbers of transmembrane helices as predicted by the TMHMM and 

SOSUI servers for the 177 membrane-integral proteins identified in the cilia preparation. 

olfactory cilia. This could be true for several kinases, calciumbinding 

proteins, or other proteins involved in signal transduction 

processes (see Supporting Information Table S2). 

Nevertheless, the high percentage of membrane proteins is a 

promising result as we are primarily interested in those proteins 

that have been purified with the cilia membranes. The 

biochemical preparation cannot separate the ciliary plasma 

membrane from membranes located within the cilia, since 

knobs, and proximal olfactory cilia contain smooth ER, multivesicular 

bodies, and other vesicular structures [14, 22, 43]. 

3.3 Functional groups in the olfactory cilia proteome 

Several functional groups of proteins are over-represented in 

the olfactory cilia proteome. 80% of the proteins detected can 

be attributed to one specific olfactory molecular task of 

ORNs. Almost one quarter (77, Fig. 4B, Supporting Information 

Table S2) of the identified proteins are related to 

intracellular traffic, consistent with the polarized protein 

expression pattern in these ciliated neurons. Proteins 

involved in cell differentiation (including ciliogenesis) as 

well as cell structure and motility are also prominent (80, 

Fig. 4B, see also groups neurogenesis and cytoskeleton in 

Supporting Information Table S2), reflecting the continuous 

turnover of ORNs throughout life. Proteins involved in 

xenobiotic metabolism as well as some stress-induced proteins 

(49, Fig. 4B and Supporting Information Table S2) are 

also enriched in the cilia proteome. This collection of cytoprotective 

proteins reflects the precarious situation of a sensory 

neuron which is exposed to every environmental compound 

present in the inhaled air; these proteins protect the 

ORNs from cytotoxic compounds and terminate the sensory 

response. Several other functional categories of interest have 

been identified such as an assortment of transport proteins 

(34), proteins involved in signal transduction processes (78), 

or proteins of unknown function (30) (Fig. 4B and Supporting 

Information Table S2). Within the signal transduction 

category, all known components of the canonical olfactory 

signal-transduction cascade (25 proteins) were robustly 

identified. The high identification scores of these proteins 

are in good accordance with the immunoblot analysis and 

further corroborate the view that the cilia preparation represents 

a material in which ciliary membrane proteins are 

efficiently purified. 


Proteomics 2009, 9, 322–334 329 

Figure 4. Subcellular location of all proteins identified and functional classification of proteins in the olfactory cilia proteome. (A) All 463 

proteins of the cilia preparation were assigned to a subcelluar compartment using the Subcellular Localization Database Locate and literature 

searches. The white sections represent cytosolic proteins, light-gray sections are membrane integral proteins, dark-gray are membrane-associated 

proteins. Cytoskeletal proteins were grouped with membrane-associated proteins. (B) Functional groups of the 377 proteins 

in the olfactory cilia proteome. Annotations were deduced from electronic annotation and extensive literature searches (see Supporting 

Information Table S1). Numbers in parentheses indicate the number of proteins in each section together with the percentages 

relative to the total number of 463 or 377 proteins, respectively. 



3.4 Using the olfactory cilia proteome 

The list of ciliary proteins provided in this study can be useful 

to promote research in various fields of olfactory function. 

The “Ca 21 /K 1 -shock” deciliation protocol and the subsequent 

purification yield a material which contains all 

known components of the ciliary transduction chain. To 

examine the function of a novel protein that is listed in the 

cilia proteome, the first step would be to look at its subcellular 

expression pattern using immunohistochemistry. In 

the following, we briefly illustrate this initial investigation 

for four proteins representing distinct aspects of olfactory 

function. (i) Plexin B2: semaphorin signaling plays an 

important role in neuronal development, axon migration, 

and neuronal apoptosis [44–46]. The semaphorin receptor 

Plexin B2 has a high score in the cilia proteome and displays 

an interesting immunosignal (Fig. 2A). A subpopulation of 

ORNs express Plexin B2 from soma to dendritic knob, suggesting 

that these cells are related to the continuous process 

of neurogenesis and apoptosis that is characteristic for the 

olfactory epithelium. (ii) Flotillin 1: there is evidence that 

lipid-raft microdomains exist in olfactory cilia and concentrate 

key transduction enzymes like AC III and G aolf [47]. 

Consistent with these data, the lipid-raft-associated protein 

flotillin 1 [48] features prominently in the cilia proteome and 

is expressed at the ciliary surface of the olfactory epithelium 

(Fig. 5B). (iii) CLIC 6: a critical missing link in the olfactory 

signal transduction chain is a calcium-activated chloride 

channel that conducts most of the receptor current [3, 4]. The 

molecular identity of this channel has not yet been established 

[3–5]. A possible new candidate for this protein may be 

CLIC 6 [49, 50], a chloride channel from the cilia proteome 

which is expressed in the cilia (Fig. 5C). (iv) SLC4A1: finally, 

chloride uptake into olfactory cilia is currently an important 

research topic [6–7]. The Cl 2 /HCO 3 2 exchanger SLC4A1, 

which has a high score in the cilia proteome and appears to 

be selectively expressed in the cilia (Fig. 5D, compare to the 

expression of AC III, Fig. 5E) seems to be a promising candidate 

for a ciliary chloride-uptake pathway. These few 

examples illustrate that many proteins of the cilia proteome, 

which hitherto were not linked to olfactory function, may 

now attract attention in the respective field of olfactory research. 

4 Discussion 

Organellar proteomics has greatly advanced our understanding 

of cellular function in recent years as it provides the 

specific protein inventory of a distinct functional unit within 

the cell. For instance, the recent proteomic characterization 

of synaptic vesicles [51, 52] has pointed to numerous novel 

approaches to studying synaptic transmission. In fact, the 

vision that sees organellar proteomics as a platform for systems 

biology is being supported by an increasing number of 

studies not only on structures common to all eukaryotic cells 

(reviews: [53–55]), but particularly on key subcellular compartments 

which play the decisive role for a specific cell type. 

Examples for such key structures are synaptic vesicles in 

neurons [52], sensory outer segments in photoreceptors [41], 

and cilia in airway epithelia [56]. In the present study, we 

have analyzed the proteome of the key structure of ORNs, the 

chemosensory cilia. Since a highly enriched cilia preparation 

was a prerequisite for this project, we improved the deciliation 

process and monitored the purification of cilia through 

the enrichment of ciliary marker proteins AC III and 

CNGA4. Using a Ca 21 /K 1 -shock method for deciliation, we 

achieved a roughly 100-fold enrichment of ciliary material. 

The cactus-like appearance of the dendritic endings of ORNs 

at the deciliated surface suggests that the cilia were detached 

at the thick proximal segments (about 2 mm long; [35]) so 

that the thin distal segments (about 50 mm long) were predominantly 

collected in the membrane preparation. Mammalian 

olfactory cilia are immotile and have accordingly 

simple axonemal structures without dynein arms. While the 

proximal segments show nine outer tubulin doublets plus 

two central subfibers, the outer doublets are missing in the 

distal cilia (review: [35]). The contribution of axonemal proteins 

to our cilia preparation is, therefore, not as prominent 

as in preparations from motile cilia (see [40]). Nevertheless, 

Figure 5. Immunohistochemical localization of four novel proteins from the cilia proteome. Cryosections of rat olfactory epithelium were 

stained with antibodies raised against the indicated proteins. The selected proteins are examples for proteins which were hitherto not in 

the focus of olfactory research but may be interesting for the fields of olfactory neurogenesis (plexin B2, A), lipid-raft microdomains (flotillin 

1, B), chemo-electrical transduction (CLIC 6, C), and ciliary chloride transport (SLC4A1, D). The immune fluorescence of AC III (E) serves 

as an example for a restricted ciliary expression. Blue signal are DAPI stains of nuclei. Calibration bars indicate 8 mm. 


Proteomics 2009, 9, 322–334 331 

51 proteins could be attributed to the cytoskeleton indicating 

that these proteins play a crucial role in providing structural 

stability and in organizing the proper targeting of membrane 

components in the olfactory cilia (see below). 

Contamination from nonciliary material may originate 

from the dendrites or somata of ORNs as well as from epithelial 

supporting cells. However, our deciliation method 

was designed to maintain tissue integrity, and SEM and 

light-microscopic inspection confirmed that the deciliated 

tissue was not noticeably damaged. Moreover, the most 

exposed epithelial structures, the supporting cell microvilli, 

were apparently resistant to the Ca 21 /K 1 -shock treatment, as 

demonstrated by the ezrin-immunohistochemistry and the 

low ezrin content of the cilia preparation. Nevertheless, contaminating 

nonciliary proteins are present in the preparation 

and have to be distinguished from ciliary proteins by bioinformatic 

analysis. 

Inspection of Supporting Information Table S2 reveals 

some interesting features of the ORN cilia proteome: The cilia 

proteome contains all proteins involved in the current model 

of chemoelectrical transduction (AC III, G-proteins, PDE1C, 

CNG channel subunits, etc.; review: [1]) with the exception of 

the OR proteins. The absence of identified ORs is not surprising, 

as each of the ,1300 members of this largest protein 

family is expressed in only about 0.1% of ORNs, thus contributing 

only very little to the cilia proteome of all ORNs. In 

the group of transduction proteins, additional 63 proteins 

were listed that have not previously been identified or investigated 

in olfactory sensory signal transduction. Among them, 

eight annexin isoforms were detected. Annexins belong to a 

class of membrane-associated Ca 21 - and phospholipid-binding 

proteins and mediate cellular responses to changes of the 

intracellular Ca 21 level. They are generally thought to act 

either as ion channel regulators or as ion channels themselves 

[57]. Their presence may point to regulatory effects on olfactory 

transduction channels. Recently, immunohistochemical 

analysis revealed that annexin isoforms A1, A2, and A5 localize 

in frog olfactory cilia [58]. Interesting proteins in this 

group also include lipid-raft associated flotillins and stomatinlike 

proteins. Stomatin-like protein-3 (SLP3) is specifically 

expressed in the cilia of ORNs and has been found to be associated 

with olfactory transduction components (e.g., AC III) 

[59, 60]. SLP3 has recently also been shown to be a constituent 

of mammalian cutaneous mechanoreceptors where it is necessary 

for the function of the mechano-sensitive transduction 

channel [61]. Thus, SLP3 is a very interesting protein, apparently 

contributing to ion-channel function in sensory neurons. 

The same could be true for the hitherto unknown stomatin-like 

member 2 in ORNs. 

Membrane-transport proteins were particularly abundant. 

The ciliary membrane separates the ORN cytosol from 

the olfactory mucus, a medium with a characteristic ionic 

composition, distinct from intra- and extracellular fluids [62]. 

The cilia proteome contains 34 ion channel or transport 

proteins of different protein families. Among them, 13 

solute transporters from nine SLC families were found, 

including SLC 12-A2, the Na 1 /K 1 /2Cl 2 cotransporter 

NKCC1 which plays a central role in olfactory transduction. 

NKCC1 accumulates intracellular Cl 2 in ORNs and provides 

the ionic basis for depolarizing Cl 2 currents [6–8]. All three 

subunits of the cAMP-gated cation channel were detected, 

together with various chloride channels, different ion 

pumps, water channels, and a set of transporters for larger 

molecules. The plasma membrane Ca 21 ATPase PMCA has 

recently been shown to contribute to re-establish resting 

Ca 21 levels in the cilia following olfactory responses [63]. The 

physiological significance of the transport capacity of cilia 

will be interesting to examine. Likely functions are the ionic 

homeostasis in the ciliary lumen and in the mucus layer. A 

great number of proteins could be classified as being 

involved in protein targeting and neurogenesis. These are 

obviously two aspects of critical importance for the olfactory 

neuroepithelium, where the neuron population is subjected 

to a constant turnover, and where a distinct transduction 

compartment is maintained by polarized expression of proteins 

involved in sensory signal processing. Accordingly, we 

found many proteins involved in the secretory transport 

machinery. Among them, receptor-transporting protein 1 

(RTP1) was shown to be specifically expressed in ORNs and 

to promote functional cell surface expression of ORs when 

expressed in HEK293T cells [17]. RTP1 is directly associated 

with the OR protein and thus also enhances the responses to 

odorants. Similar results were found for HSC70 [64]. Understanding 

OR function is hampered by the difficulty in heterologous 

expression. The proteins listed in this category are 

good olfactory candidates to enhance further experimental 

approaches in this problem. Alternatively, they could also 

contribute to the targeting of other signal transduction 

molecules to the ciliary membrane. In particular, Lambert et 

al. [65] showed that B-cell receptor-associated protein 31 

(BAP31) colocalizes with and controls the expression of cystic 

fibrosis transmembrane conductance regulator (CFTR) in 

the plasma membrane. Similar findings were reported for 

several RAB isoforms present in the cilia proteome [66]. 

Chen and Balch [67] reported that RAB recycling in the early 

secretory pathways involves the heat-shock protein HSP90 

system. HSP90 activity is required to form a functional guanine 

nucleotide dissociation inhibitor complex to retrieve, 

e.g., RAB1 from the membrane and is essential for RAB1- 

dependent Golgi assembly. The three HSP90 proteins identified 

in our analysis seem to be involved in the RAB pathway. 

Moreover, we found several proteins involved in neurogenesis. 

For example, the neuronal marker protein growth associated 

protein (GAP-43) is expressed in olfactory neurons 

during growth [68]. KPL2 is an actin-binding protein, 

induced during ciliogenesis [56], and may be involved in the 

differentiation of ORNs from precursor cells. The same 

could be true for several other proteins listed in this group. 

The substantial number of identified olfactory cytoprotective 

proteins reflects the special location of ORNs as the only 

neurons of the body directly exposed to the external environment. 



The 377 proteins identified here do not constitute the 

entire cilia proteome. Proteins with low abundancy which 

did not reach the identification threshold (see Section 2) 

include the olfactory receptor proteins, and probably also 

membrane proteins expressed in small subpopulations of 

ORNs in the olfactory epithelium. Improved methods of 

membrane isolation are needed for a more specific purification 

of membrane proteins. The selective partitioning of 

plasma membranes in aqueous polymer two-phase systems 

is a promising approach to this problem [69]. This 

method exploits the tendency of biological membranes to 

partition into the top phase of a poly ethylene glycol/dextran 

system from where a high fraction of plasma membrane 

proteins can be isolated. Such a protocol reduces the 

complexity of protein mixtures in favor of plasma membrane 

proteins and may give access to low-abundancy 

proteins. 

In conclusion, we have isolated sensory cilia of rat 

ORN by a novel deciliation method. The quality of the 

preparation was monitored by SEM, ciliary marker proteins, 

and bioinformatic examination. The resulting list of 

377 proteins contains most of the known proteins of 

olfactory signal transduction, as well as many other proteins 

that may fulfill important functions in olfactory 

physiology. The cilia proteome provides multiple starting 

points for more detailed examinations of distinct protein 

functions in ORNs. Especially for the areas of membrane 

transport, signal processing, membrane targeting, and 

neurogenesis, the cilia proteome constitutes a valuable 

source of information. 

We are grateful to Dr. Tore Kempf (DKFZ Heidelberg) for 

valuable help with the protein analysis and Georgia Ignatiadou 

(Institute of Zoology, University of Heidelberg) for technical 

assistance with the immunohistochemistry. 

The authors have declared no conflict of interest. 

5 References 

[1] Frings, S., Chemoelectrical signal transduction in olfactory 

sensory neurons of air-breathing vertebrates. Cell Mol. Life 

Sci. 2001, 58, 510–519. 

[2] Hatt, H., Molecular and cellular basis of human olfaction. 

Chem. Biodivers. 2004, 1, 1857–1869. 

[3] Kleene, S. J., Gesteland, R. C., Calcium-activated chloride 

conductance in frog olfactory cilia. J. Neurosci. 1991, 11, 

3624–3629. 

[4] Reisert, J., Bauer, P. J., Yau, K. W., Frings, S., The Ca-activated 

Cl channel and its control in rat olfactory receptor neurons. J. 

Gen. Physiol. 2003, 122, 349–363. 

[5] Pifferi, S., Pascarella, G., Boccaccio, A., Mazzatenta, A. et al., 

Bestrophin-2 is a candidate calcium-activated chloride channel 

involved in olfactory transduction. Proc. Natl. Acad. Sci. 

USA 2006, 103, 12929–12934. 

[6] Kaneko, H., Putzier, I., Frings, S., Kaupp, U. B., Gensch, T., 

Chloride accumulation in mammalian olfactory sensory 

neurons. J. Neurosci. 2004, 24, 7931–7938. 

[7] Reisert, J., Lai, J., Yau, K. W., Bradley, J., Mechanism of the 

excitatory Cl- response in mouse olfactory receptor neurons. 

Neuron 2005, 45, 553–561. 

[8] Nickell, W. T., Kleene, N. K., Kleene, S. J., Mechanisms of 

neuronal chloride accumulation in intact mouse olfactory 

epithelium. J. Physiol. 2007, 583, 1005–1020. 

[9] Smith, D. W., Thach, S., Marshall, E. L., Mendoza, M. G., 

Kleene, S. J., Mice lacking NKCC1 have normal olfactory 

sensitivity. Physiol. Behav. 2008 ,93, 44–49. 

[10] Boekhoff, I., Inglese, J., Schleicher, S., Koch, W. J. et al., 

Olfactory desensitization requires membrane targeting of 

receptor kinase mediated by beta gamma-subunits of heterotrimeric 

G proteins. J. Biol. Chem. 1994, 269, 37–40. 

[11] Wei, J., Zhao, A. Z., Chan, G. C., Baker, L. P. et al., Phosphorylation 

and inhibition of olfactory adenylyl cyclase by CaM 

kinase II in Neurons: A mechanism for attenuation of olfactory 

signals. Neuron 1998, 21, 495–504. 

[12] Bradley, J., Bonigk, W., Yau, K. W., Frings, S., Calmodulin 

permanently associates with rat olfactory CNG channels 

under native conditions. Nat. Neurosci. 2004, 7, 705–710. 

[13] Boccaccio, A., Lagostena, L., Hagen, V., Menini, A., Fast 

adaptation in mouse olfactory sensory neurons does not 

require the activity of phosphodiesterase. J. Gen. Physiol. 

2006, 128, 171–184. 

[14] Mashukova, A., Spehr, M., Hatt, H., Neuhaus, E. M., Betaarrestin2-mediated 

internalization of mammalian odorant 

receptors. J. Neurosci. 2006, 26, 9902–9912. 

[15] Cowan, C. M., Roskams, A. J., Apoptosis in the mature and 

developing olfactory neuroepithelium. Microsc. Res. Tech. 

2002, 58, 204–215. 

[16] Henion, T. R., Schwarting, G. A., Patterning the developing 

and regenerating olfactory system. J. Cell Physiol. 2007, 210, 

290–297. 

[17] Saito, H., Kubota, M., Roberts, R. W., Chi, Q., Matsunami, H., 

RTP family members induce functional expression of mammalian 

odorant receptors. Cell 2004, 119, 679–691. 

[18] Michalakis, S., Reisert, J., Geiger, H., Wetzel, C. et al.,Lossof 

CNGB1 protein leads to olfactory dysfunction and subciliary 

cyclic nucleotide-gated channel trapping. J. Biol. Chem. 

2006, 281, 35156–35166. 

[19] Liberles, S. D., Buck, L. B., A second class of chemosensory 

receptors in the olfactory epithelium. Nature 2006, 442, 645– 

650. 

[20] Spehr, M., Kelliher, K. R., Li, X. H., Boehm, T. et al., Essential 

role of the main olfactory system in social recognition of 

major histocompatibility complex peptide ligands. J. Neurosci. 

2006, 26, 1961–1970. 

[21] Leinders-Zufall, T., Cockerham, R. E., Michalakis, S., Biel, M. 

et al., Contribution of the receptor guanylyl cyclase GC-D to 

chemosensory function in the olfactory epithelium. Proc. 

Natl. Acad. Sci. USA 2007, 104, 14507–14512. 

[22] Mayer, U., Ungerer, N., Klimmeck, D., Warnken, U. et al., 

Proteomic analysis of a membrane preparation from rat 

olfactory sensory cilia. Chem. Senses 2007, 33, 145–162. 

[23] Bonigk, W., Bradley, J., Muller, F., Sesti, F. et al., The native 

rat olfactory cyclic nucleotide-gated channel is composed of 

three distinct subunits. J. Neurosci. 1999, 19, 5332–5347. 


Proteomics 2009, 9, 322–334 333 

[24] Bradford, M. M., A rapid and sensitive method for the quantitation 

of microgram quantities of protein utilizing the principle 

of protein-dye binding. Anal. Biochem. 1976, 72, 248– 

254. 

[25] Laemmli, U. K., Cleavage of structural proteins during the 

assembly of the head of bacteriophage T4. Nature 1970, 227, 

680–685. 

[26] Mayer, U., Protein Information Crawler (PIC): Extensive spidering 

of multiple protein information resources for large 

protein sets. Proteomics 2008, 8, 42–44. 

[27] Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. 

J., Basic local alignment search tool. J. Mol. Biol. 1990, 215, 

403–410. 

[28] Altschul, S. F., Gish, W., Local alignment statistics. Methods 

Enzymol. 1996, 266, 460–480. 

[29] Liebel, U., Kindler, B., Pepperkok, R., ‘Harvester’: A fast meta 

search engine of human protein resources. Bioinformatics 

2004, 20, 1962–1963. 

[30] Krogh, A., Larsson, B., von Heijne, G., Sonnhammer, E. L., 

Predicting transmembrane protein topology with a hidden 

Markov model: Application to complete genomes. J. Mol. 

Biol. 2001, 305, 567–580. 

[31] Hirokawa, T., Boon-Chieng, S., Mitaku, S., SOSUI: Classification 

and secondary structure prediction system for membrane 

proteins. Bioinformatics 1998, 14, 378–379. 

[32] Fink, J. L., Aturaliya, R. N., Davis, M. J., Zhang, F. et al., 

LOCATE: A mouse protein subcellular localization database. 

Nucleic Acids Res. 2006, 34, D213–D217. 

[33] Adoutte, A., Ramanathan, R., Lewis, R. M., Dute, R. R. et al., 

Biochemical studies of the excitable membrane of Paramecium 

tetraurelia. III. Proteins of cilia and ciliary membranes. 

J. Cell Biol. 1980, 84, 717–738. 

[34] Menco, B. P., Bruch, R. C., Dau, B., Danho, W., Ultrastructural 

localization of olfactory transduction components: The G 

protein subunit Golf alpha and type III adenylyl cyclase. 

Neuron 1992, 8, 441–453. 

[35] Menco, B. P., Ultrastructural aspects of olfactory signaling. 

Chem. Senses 1997, 22, 295–311. 

[36] Elsaesser, R., Montani, G., Tirindelli, R., Paysan, J., Phosphatidyl-inositide 

signalling proteins in a novel class of 

sensory cells in the mammalian olfactory epithelium. Eur. J. 

Neurosci. 2005, 21, 2692–2700. 

[37] Bakalyar, H. A., Reed, R. R., Identification of a specialized 

adenylyl cyclase that may mediate odorant detection. Science 

1990, 250, 1403–1406. 

[38] Juilfs, D. M., Fulle, H. J., Zhao, A. Z., Houslay, M. D. et al., A 

subset of olfactory neurons that selectively express cGMPstimulated 

phosphodiesterase (PDE2) and guanylyl cyclase- 

D define a unique olfactory signal transduction pathway. 

Proc. Natl. Acad. Sci. USA 1997, 94, 3388–3395. 

[39] Sammeta, N., Yu, T. T., Bose, S. C., McClintock, T. S., Mouse 

olfactory sensory neurons express 10 000 genes. J. Comp. 

Neurol. 2007, 502, 1138–1156. 

[40] Gherman, A., Davis, E. E., Katsanis, N., The ciliary proteome 

database: An integrated community resource for the genetic 

and functional dissection of cilia. Nat. Genet. 2006, 38, 961– 

962. 

[41] Liu, Q., Tan, G., Levenkova, N., Li, T. et al., The proteome of 

the mouse photoreceptor sensory cilium complex. Mol. Cell. 

Proteomics 2007, 6, 1299–1317. 

[42] Anholt, R. R., Aebi, U., Snyder, S. H., A partially purified 

preparation of isolated chemosensory cilia from the olfactory 

epithelium of the bullfrog, Rana catesbeiana. J. Neurosci. 

1986, 6, 1962–1969. 

[43] Andres, K. H., The olfactory epithelium of the cat. Z. Zellforsch. 

Mikrosk. Anat. 1969, 96, 250–274. 

[44] Deng, S., Hirschberg, A., Worzfeld, T., Penachioni, J. Y. et al., 

Plexin-B2, but not Plexin-B1, critically modulates neuronal 

migration and patterning of the developing nervous system 

in vivo. J. Neurosci. 2007, 27, 6333–6347. 

[45] Fujisawa, H., Discovery of semaphorin receptors, neuropilin 

and plexin, and their functions in neural development. J. 

Neurobiol. 2004, 59, 24–33. 

[46] Shirvan, A., Ziv, I., Fleminger, G., Shina, R. et al., Semaphorins 

as mediators of neuronal apoptosis. J. Neurochem. 

1999, 73, 961–971. 

[47] Schreiber, S., Fleischer, J., Breer, H., Boekhoff, I., A possible 

role for caveolin as a signaling organizer in olfactory sensory 

membranes. J. Biol. Chem. 2000, 275, 24115–24123. 

[48] Babuke, T., Tikkanen, R., Dissecting the molecular function 

of reggie/flotillin proteins. Eur. J. Cell Biol. 2007, 86, 525–532. 

[49] Griffon, N., Jeanneteau, F., Prieur, F., Diaz, J., Sokoloff, P., 

CLIC6, a member of the intracellular chloride channel family, 

interacts with dopamine D(2)-like receptors. Brain Res. Mol. 

Brain Res. 2003, 117, 47–57. 

[50] Friedli, M., Guipponi, M., Bertrand, S., Bertrand, D. et al., 

Identification of a novel member of the CLIC family, CLIC6, 

mapping to 21q22.12. Gene 2003, 320, 31–40. 

[51] Takamori, S., Holt, M., Stenius, K., Lemke, E. A. et al., Molecular 

anatomy of a trafficking organelle. Cell 2006, 127, 

831–846. 

[52] Burre, J., Volknandt, W., The synaptic vesicle proteome. J. 

Neurochem. 2007, 101, 1448–1462. 

[53] Yates, J. R., III, Gilchrist, A., Howell, K. E., Bergeron, J. J., 

Proteomics of organelles and large cellular structures. Nat. 

Rev. Mol. Cell Biol. 2005, 6, 702–714. 

[54] Andersen, J. S., Mann, M., Organellar proteomics: Turning 

inventories into insights. EMBO Rep. 2006, 7, 874–879. 

[55] Au, C. E., Bell, A. W., Gilchrist, A., Hiding, J. et al., Organellar 

proteomics to create the cell map. Curr. Opin. Cell Biol. 2007, 

19, 376–385. 

[56] Ostrowski, L. E., Blackburn, K., Radde, K. M., Moyer, M. B. et 

al., A proteomic analysis of human cilia: Identification of 

novel components. Mol. Cell. Proteomics 2002, 1, 451–465. 

[57] Gerke, V., Moss, S. E., Annexins: From structure to function. 

Physiol. Rev. 2002, 82, 331–371. 

[58] Uebi, T., Miwa, N., Kawamura, S., Comprehensive interaction 

of dicalcin with annexins in frog olfactory and respiratory 

cilia. FEBS J. 2007, 274, 4863–4876. 

[59] Kobayakawa, K., Hayashi, R., Morita, K., Miyamichi, K. et al., 

Stomatin-related olfactory protein, SRO, specifically 

expressed in the murine olfactory sensory neurons. J. Neurosci. 

2002, 22, 5931–5937. 

[60] Goldstein, B. J., Kulaga, H. M., Reed, R. R., Cloning and 

characterization of SLP3: A novel member of the stomatin 

family expressed by olfactory receptor neurons. J. Assoc. 

Res. Otolaryngol. 2003, 4, 74–82. 

[61] Wetzel, C., Hu, J., Riethmacher, D., Benckendorff, A. et al., A 

stomatin-domain protein essential for touch sensation in 

the mouse. Nature 2007, 445, 206–209. 



[62] Reuter, D., Zierold, K., Schroder, W. H., Frings, S., A depolarizing 

chloride current contributes to chemoelectrical 

transduction in olfactory sensory neurons in situ. J. Neurosci. 

1998, 18, 6623–6630. 

[63] Castillo, K., Delgado, R., Bacigalupo, J., Plasma membrane 

Ca(21)-ATPase in the cilia of olfactory receptor neurons: 

Possible role in Ca(21) clearance. Eur. J. Neurosci. 2007, 26, 

2524–2531. 

[64] Neuhaus, E. M., Mashukova, A., Zhang, W., Barbour, J., Hatt, 

H., A specific heat shock protein enhances the expression of 

mammalian olfactory receptor proteins. Chem. Senses 

2006, 31, 445–452. 

[65] Lambert, G., Becker, B., Schreiber, R., Boucherot, A. et al., 

Control of cystic fibrosis transmembrane conductance regulator 

expression by BAP31. J. Biol. Chem. 2001, 276, 20340– 

20345. 

[66] Saxena, S. K., Kaur, S., Regulation of epithelial ion channels 

by Rab GTPases. Biochem. Biophys. Res. Commun. 2006, 

351, 582–587. 

[67] Chen, C. Y., Balch, W. E., The Hsp90 chaperone complex 

regulates GDI-dependent Rab recycling. Mol. Biol. Cell 2006, 

17, 3494–3507. 

[68] Weiler, E., Benali, A., Olfactory epithelia differentially 

express neuronal markers. J. Neurocytol. 2005, 34, 217–240. 

[69] Schindler, J., Nothwang, H. G., Aqueous polymer two-phase 

systems: Effective tools for plasma membrane proteomics. 

Proteomics 2006, 6, 5409–5417.

The proteome of rat olfactory sensory cilia

Create successful ePaper yourself

Delete template?

Save as template?