A previously unrecognized membrane protein in the Rhodobacter sphaeroides LH1-RC photocomplex

AbstractRhodobacter (Rba.) sphaeroides is the most widely used model organism in bacterial photosynthesis. The light-harvesting-reaction center (LH1-RC) core complex of this purple phototroph is characterized by the co-existence of monomeric and dimeric forms, the presence of the protein PufX, and approximately two carotenoids per LH1 αβ-polypeptides. Despite many efforts, structures of the Rba. sphaeroides LH1-RC have not been obtained at high resolutions. Here we report a cryo-EM structure of the monomeric LH1-RC from Rba. sphaeroides strain IL106 at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its channels. Our findings offer an exciting new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

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A Novel Membrane Protein in the Rhodobacter sphaeroides LH1-RC Photocomplex

10.1101/2021.06.20.449127 ◽

2021 ◽

Author(s):

K. Tani ◽

K. V. P. Nagashima ◽

R. Kanno ◽

S. Kawamura ◽

R. Kikuchi ◽

...

Keyword(s):

Membrane Protein ◽

Ring Opening ◽

Hypothetical Protein ◽

Integral Membrane Protein ◽

Purple Bacterium ◽

Core Complex ◽

Rc Structures ◽

Density Map ◽

Complex Forms ◽

And Function

We present a cryo-EM structure of the monomeric light-harvesting-reaction center (LH1-RC) core complex from photosynthetic purple bacterium Rhodobacter (Rba.) sphaeroides at 2.9 Å resolution. The LH1 complex forms a C-shaped structure composed of 14 αβ-polypeptides around the RC with a large ring opening. From the cryo-EM density map, a previously unrecognized integral membrane protein, referred to as protein-U, was identified. Protein-U has a U-shaped conformation near the LH1-ring opening and was annotated as a hypothetical protein in the Rba. sphaeroides genome. Deletion of protein-U resulted in a mutant strain that expressed a much-reduced amount of the dimeric LH1-RC, indicating an important role for protein-U in dimerization of the LH1-RC complex. PufX was located opposite protein-U on the LH1-ring opening, and both its position and conformation differed from that of previous reports of dimeric LH1-RC structures obtained at low-resolution. Twenty-six molecules of the carotenoid spheroidene arranged in two distinct configurations were resolved in the Rba. sphaeroides LH1 and were positioned within the complex to block its pores. Our findings offer a new view of the core photocomplex of Rba. sphaeroides and the connections between structure and function in bacterial photocomplexes in general.

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APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

10.1101/2020.03.09.984815 ◽

2020 ◽

Author(s):

Daniel E. Vélez-Ramírez ◽

Michelle M. Shimogawa ◽

Sunayan Ray ◽

Andrew Lopez ◽

Shima Rayatpisheh ◽

...

Keyword(s):

Membrane Protein ◽

Trypanosoma Brucei ◽

Model Organism ◽

Protein Composition ◽

Principal Component ◽

Protozoan Parasite ◽

Sleeping Sickness ◽

Critical Gap ◽

Flagellar Membrane ◽

And Function

ABSTRACTTrypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

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The yeast integral membrane protein Apq12 potentially links membrane dynamics to assembly of nuclear pore complexes

The Journal of Cell Biology ◽

10.1083/jcb.200702120 ◽

2007 ◽

Vol 178 (5) ◽

pp. 799-812 ◽

Cited By ~ 63

Author(s):

John J. Scarcelli ◽

Christine A. Hodge ◽

Charles N. Cole

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

Membrane Dynamics ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Cold Sensitive ◽

Low Levels ◽

Lower Temperature ◽

Pore Complexes ◽

And Function

Although the structure and function of components of the nuclear pore complex (NPC) have been the focus of many studies, relatively little is known about NPC biogenesis. In this study, we report that Apq12 is required for efficient NPC biogenesis in Saccharomyces cerevisiae. Apq12 is an integral membrane protein of the nuclear envelope (NE) and endoplasmic reticulum. Cells lacking Apq12 are cold sensitive for growth, and a subset of their nucleoporins (Nups), those that are primarily components of the cytoplasmic fibrils of the NPC, mislocalize to the cytoplasm. APQ12 deletion also causes defects in NE morphology. In the absence of Apq12, most NPCs appear to be associated with the inner but not the outer nuclear membrane. Low levels of benzyl alcohol, which increases membrane fluidity, prevented Nup mislocalization and restored the proper localization of Nups that had accumulated in cytoplasmic foci upon a shift to lower temperature. Thus, Apq12p connects nuclear pore biogenesis to the dynamics of the NE.

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Topology and function of the integral membrane protein conferring immunity to colicin A

Molecular Microbiology ◽

10.1111/j.1365-2958.1989.tb00216.x ◽

1989 ◽

Vol 3 (5) ◽

pp. 679-687 ◽

Cited By ~ 36

Author(s):

V. Geli ◽

D. Baty ◽

F. Pattus ◽

C. Lazdunski

Keyword(s):

Membrane Protein ◽

Integral Membrane Protein ◽

And Function

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Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5671 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5671-5681 ◽

Cited By ~ 281

Author(s):

P Mazur ◽

N Morin ◽

W Baginsky ◽

M el-Sherbeini ◽

J A Clemas ◽

...

Keyword(s):

Membrane Protein ◽

Function Analysis ◽

Calcineurin Inhibitors ◽

Integral Membrane Protein ◽

Dependent Manner ◽

Fungal Cell ◽

Glucan Synthase ◽

Increased Sensitivity ◽

And Function ◽

Null Mutants

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p. The residual glucan synthase activity present in strains with deletions of fks1 is (i) immunodepleted by antibodies prepared against FKS2 peptides, demonstrating that Fks2p is also a component of the enzyme, and (ii) more sensitive to the echinocandin L-733,560, explaining the increased sensitivity of fks1 null mutants to this drug. Simultaneous disruption of FKS1 and FKS2 is lethal, suggesting that Fks1p and Fks2p are alternative subunits with essential overlapping function. Analysis of FKS1 and FKS2 expression reveals that transcription of FKS1 is regulated in the cell cycle and predominates during growth on glucose, while FKS2 is expressed in the absence of glucose. FKS2 is essential for sporulation, a process which occurs during nutritional starvation. FKS2 is induced by the addition of Ca2+ to the growth medium, and this induction is completely dependent on the Ca2+/calmodulin-dependent phosphoprotein phosphatase calcineurin. We have previously shown that growth of fks1 null mutants is highly sensitive to the calcineurin inhibitors FK506 and cyclosporin A. Expression of FKS2 from the heterologous ADH1 promoter results in FK506-resistant growth. Thus, the sensitivity of fks1 mutants to these drugs can be explained by the calcineurin-dependent transcription of FKS2. Moreover, FKS2 is also highly induced in response to pheromone in a calcineurin-dependent manner, suggesting that FKS2 may also play a role in the remodeling of the cell wall during the mating process.

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Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: Application to phospholamban

Biopolymers ◽

10.1002/bip.10365 ◽

2003 ◽

Vol 69 (3) ◽

pp. 283-292 ◽

Cited By ~ 25

Author(s):

Nathan A. Lockwood ◽

Raymond S. Tu ◽

Zhiwen Zhang ◽

Matthew V. Tirrell ◽

David D. Thomas ◽

...

Keyword(s):

Membrane Protein ◽

Protein Domains ◽

Integral Membrane Protein ◽

Structure And Function ◽

Peptide Amphiphiles ◽

And Function

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The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

Molecular Biology of the Cell ◽

10.1091/mbc.9.2.355 ◽

1998 ◽

Vol 9 (2) ◽

pp. 355-373 ◽

Cited By ~ 33

Author(s):

Albert K. Ho ◽

Gregory A. Raczniak ◽

Eric B. Ives ◽

Susan R. Wente

Keyword(s):

Membrane Protein ◽

Transmembrane Domain ◽

Integral Membrane Protein ◽

Genetic Screen ◽

Terminal Region ◽

Yeast Cells ◽

Nuclear Pore ◽

Endoplasmic Reticulum Membrane ◽

Carboxyl Terminal ◽

And Function

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered thenup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethalnup116-C phenotype was conducted. One gene (designatedSNL1 for suppressor of n up116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity ofgle2–1 and nic96-G3 mutant cells. Thenic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporinnup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

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Self-Interaction Is Critical for Atg9 Transport and Function at the Phagophore Assembly Site during Autophagy

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0544 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5506-5516 ◽

Cited By ~ 77

Author(s):

Congcong He ◽

Misuzu Baba ◽

Yang Cao ◽

Daniel J. Klionsky

Keyword(s):

Membrane Protein ◽

Membrane Transport ◽

Integral Membrane Protein ◽

Human Diseases ◽

Anterograde Transport ◽

Membrane Flow ◽

Phagophore Assembly Site ◽

And Function ◽

Assembly Site ◽

Starvation Conditions

Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and is proposed to play a key role in membrane transport; the phagophore presumably expands by vesicular addition to form a complete autophagosome. It is not clear through what mechanism Atg9 functions at the phagophore assembly site (PAS). Here we report that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses reveal that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Our results support a model in which Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation.

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