Palmitoylated proteins: purification and identification

Wan, Junmei; Roth, Allan C.; Bailey, Aaron O.; Davis, Nicholas G.

doi:10.1038/nprot.2007.225

Cited by 385 publications

(404 citation statements)

References 20 publications

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“…With poorly defined palmitoylation motifs (41), proteomic strategies (15,42) will be required to identify the true extent of protein palmitoylation in these stages, characterize their effect on parasite morphogenesis, and determine whether crystalloid formation and maintenance necessitates palmitoylation of crystalloid-resident proteins such as the LAPs. The identification of a clear phenotype linked to DHHC10 will help define enzyme-substrate pairs.…”

Section: Discussionmentioning

confidence: 99%

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

Santos

Duarte

Kehrer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogonyis dependent on the precisely timed expression of the S-acyltransferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission.T he malaria parasite is capable of infecting both the vertebrate host and mosquito vector. After a mosquito blood meal, sexual precursor cells rapidly differentiate into mature gametes. In the mosquito midgut, the gametes mate to form a zygote that develops further into the motile ookinete. After crossing the midgut epithelium and establishing a sessile oocyst, the ookinete gives rise to thousands of sporozoites capable of infecting a subsequent mammalian host (1).Sharing key organelles like the nucleus, endoplasmic reticulum, Golgi, and mitochondria with other eukaryotes, this parasite has evolved specialized, stage-specific structures that are necessary for developmental progression during parasite transmission. These include, for example, osmiophilic bodies (secretory vesicles) that release protein factors capable of lysing the parasitophorous vacuole and erythrocyte membranes, thus producing free gametes (2) and a gliding motility motor anchored to the inner membrane complex (IMC), allowing the ookinete to migrate across the mosquito midgut epithelium and establish an oocyst (3). Sporozoite formation in the oocyst finally requires the presence of a stage-specific organelle, the crystalloid, a multivesicular structure assembled in the ookinete and putative reservoir of proteins and lipids used during sporogony. Although this enigmatic organelle was discovered more than 40 y ago, its formation and function remain largely unknown (4-9). Six LCCL proteins have been shown to reside within (9) and maintain the stability (8, 9) of these organelles essential for sporogony (10).The morphological changes taking place during zygote-toookinete development and the generation of thousands of sporozoites inside a single oocyst require extensive protein translation and membrane biogenesis to support the formation of organelles and plasma membrane (PM) surrounding each new parasite. Onethird of the proteins identified in the oocyst and oocyst-derived (midgut) sporozoites of the human parasite Plasmodium falciparum are putatively membrane-bound (11). The targeting of such proteins to organelles, and perhaps formation of certain organelles per se, requires appropriate sorting signals, along with transmembrane (TM) domains to keep these factors in place. Posttranslational modifications, such as lipidation, can increase the affinity of a modif...

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Section: Discussionmentioning

confidence: 99%

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

Santos

Duarte

Kehrer

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Second, to confirm the predicted palmitoylation in vivo, parasites expressing either the wild type sequence ( 20 PfISP3-GFP and 20 PfISP1-GFP) or the corresponding cysteine/alanine substitutions ( 20 PfISP3 C5AC6A -GFP and 20 PfISP1 C7AC8A -GFP) were used in acyl biotin exchange assays (21,58). This assay substitutes thioester-linked acyl groups with biotin that can subsequently be used for affinity purification.…”

Section: Journal Of Biological Chemistrymentioning

confidence: 99%

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

Wetzel

Herrmann

Swapna

et al. 2015

Journal of Biological Chemistry

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Background: Recruitment of peripheral proteins to the inner membrane complex (IMC) of the malaria parasite can be mediated by N-terminal acylation. Results: Characterization of substrate determinants and identification of an IMC-localized palmitoyl acyltransferase PfDHHC1. Conclusion: Residues close to palmitoylation sites interfere with specific IMC recruitment. PfDHHC1 represents an apicomplexan-specific PAT. Significance: Dissection of palmitoylation for protein recruitment to the inner membrane complex in P. falciparum.

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“…Distinct protein bands were cut out from gels, digested with trypsin according to a published protocol (20), and analyzed by mass spectrometry at the Center for Proteomics and Bioinformatics at Case Western Reserve University. Alternatively, fractions containing purified RPE65 and its cross-linked complexes were pooled together, and the proteins were precipitated by a chloroform/ methanol procedure (21). Protein pellets were resuspended in 5 l of 50% formic acid in water; the acid was neutralized with 0.2 ml of 200 mM ammonium bicarbonate, and the proteins were digested with sequence grade trypsin overnight at 37°C.…”

Section: Purification Of Cross-linked Products and Identification Of mentioning

confidence: 99%

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

Golczak

Kiser

Lodowski

et al. 2010

Journal of Biological Chemistry

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Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A 2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.In vertebrates, the perception of light is preceded by a quantum event in which a photon of light hits the 11-cis-retinylidene chromophore of rhodopsin in the retina. Photoisomerization of this chromophore to its all-trans isomer results in activation of rhodopsin triggering a cascade of signal transduction events (summarized in Ref. 1). This process simultaneously leads to loss of photoreceptor light sensitivity making continuous regeneration of 11-cis-retinal a requirement to sustain vision. The visual (retinoid) cycle is the fundamental series of reactions responsible for regeneration of the visual chromophore that occurs in photoreceptors and the retinal pigment epithelium (RPE) 4 (summarized in Ref.2). The key component of this enzymatic cycle is the RPE-specific microsomal membrane protein known as RPE65 (3-5). The importance of RPE65 in the retinoid cycle is underscored by the metabolic dysfunction found in Rpe65 Ϫ/Ϫ mice, which is characterized by complete lack of 11-cis-retinoid production accompanied by accumulation of the all-tr...

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Palmitoylated proteins: purification and identification

Cited by 385 publications

References 20 publications

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite

The Role of Palmitoylation for Protein Recruitment to the Inner Membrane Complex of the Malaria Parasite

Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

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