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Detergent-resistant membranes in Agrobacterium tumefaciens

ABSTRACT: We established a protocol for the biochemical fractionation of Agrobacterium tumefaciens membranes, and characterized the proteome of detergent-sensitive and detergent-resistant membrane fractions by mass spectrometry. We found that well-established microdomain marker proteins co-purify with proteins associated with transport or secretion processes in the detergent-resistant fraction, suggesting a functional micro-compartmentalization of the Gram-negative membrane. The presence of components of virulence-associated secretion systems in detergent-resistant membranes supports the involvement of bacterial microdomains in host-microbe interaction and microbial competition.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Agrobacterium Fabrum Str. C58

SUBMITTER: Simon Czolkoss

LAB HEAD: Franz Narberhaus

PROVIDER: PXD028782 | Pride | 2021-11-03

REPOSITORIES: Pride

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Project description:Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1, which is a depalmitoylation enzyme result in early onset neurodegeneration, therefore, we hypothesized that the protein content of brain membranes from mutant newborn mice will differ from those derived from wild-type. This unbiased analysis revealed that the majority of the differentially identified proteins are palmitoylated and their upstream pathway analysis point to neurodegenerative diseases. One of the differentially expressed proteins was Rab3IP, which is a direct downstream effector of Rab11 and affects the guanine nucleotide-exchange activity of Rab3IP toward Rab8. Rab3IP, Rab11 and Rab8 are known to be involved in ciliogenesis. Rab3IP distribution in cilia differed between the wild type and the mutant cells. Our analysis showed that these three proteins are palmitoylated. Site-directed mutagenesis of the palmitoylation sites of Rab11 affects its intracellular localization. Most importantly, in MEFs, neuroblasts, neurons and brain preparations, longer cilia were detected in Ppt1-/-. Collectively, our studies suggest that cilia abnormalities need to be considered as part of the pathophysiology of CLN1. Mutations in Ppt1 result in early onset neurodegeneration, therefore, we hypothesized that the levels of proteins extracted from purified brain membranes of newborn mice will differ. One of the main effects of palmitoylation is changing the hydrophobic index of proteins and thus affecting these proteins interactions with different membranal domains. Assuming that PPT1 substrates are over palmitoylated in Ppt1-/- brains it is possible that its localization to specific membranal domain is different than in the wild-type brains. We isolated Triton X-100 soluble membranes from three wild-type and three Ppt1-/- brains and identified the proteins by PalmPISC. Many of the identified proteins were found to be palmitoylated. A second feature, which was noted is that many of the differential proteins (62 out of 88, 70%) can be detected in the cilia proteome (http://v3.ciliaproteome.org/cgi-bin/index.php). This observation led to test whether ciliary proteins differ between wild-type and Ppt1-/-. Cilia were prepared from newborn dissociated brain cell cultures and analyzed by MS

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Detergent-resistant membranes in Agrobacterium tumefaciens

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