The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome ... more The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome segregation by promoting error correction and spindle assembly checkpoint (SAC) satisfaction. CLASPs are widely conserved microtubule plus-end-tracking proteins that regulate microtubule dynamics throughout the cell cycle and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore microtubule dynamics, but the underlying molecular mechanism remains unknown. Here we found that human CLASP2 can dimerize through its C-terminal kinetochore-targeting domain, but kinetochore localization was independent of dimerization. CLASP2 kinetochore localization, microtubule plus-end-tracking and microtubule lattice binding through TOG2 and TOG3 (but not TOG1) domains, independently sustained normal spindle length, timely SAC satisfaction, chromosome congression and faithful segregation. Measurements of kinetochore microtubule half-life in living...
The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nu... more The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic rib...
CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophe... more CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore–microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore–microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB–protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromos...
We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of co... more We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as t...
PEX1 and PEX6 are two members of the ATPases Associated with diverse cellular Activities (AAA) fa... more PEX1 and PEX6 are two members of the ATPases Associated with diverse cellular Activities (AAA) family and the core components of the receptor export module (REM) of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex which unfolds substrates by processive threading. However, whether the natural substrate of the PEX1.PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity crosslinking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through it...
Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell... more Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized (35)S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be ...
In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to t... more In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.
Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 th... more Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interact...
Post-translational modifiers such as the Small Ubiquitin-like Modifier (SUMO) peptide act as fast... more Post-translational modifiers such as the Small Ubiquitin-like Modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has uncovered the key regulatory role that SUMO plays in plant development. Unlike SUMO conjugation pathway components, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. The present study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, uncovering the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an understudied subgroup, Arabidopsis SPF1 and SPF2 were subsequently subjected to functional characterization. Here, loss-of-function mutants implicated both proteins with vegetative growth, flowering time and seed size and yield. Mutants constitutively accumulated SUMO-conjugates, and yeast complementation assays assoc...
The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome ... more The fine regulation of kinetochore microtubule dynamics during mitosis ensures proper chromosome segregation by promoting error correction and spindle assembly checkpoint (SAC) satisfaction. CLASPs are widely conserved microtubule plus-end-tracking proteins that regulate microtubule dynamics throughout the cell cycle and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore microtubule dynamics, but the underlying molecular mechanism remains unknown. Here we found that human CLASP2 can dimerize through its C-terminal kinetochore-targeting domain, but kinetochore localization was independent of dimerization. CLASP2 kinetochore localization, microtubule plus-end-tracking and microtubule lattice binding through TOG2 and TOG3 (but not TOG1) domains, independently sustained normal spindle length, timely SAC satisfaction, chromosome congression and faithful segregation. Measurements of kinetochore microtubule half-life in living...
The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nu... more The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic rib...
CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophe... more CLASPs are conserved microtubule plus-end–tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore–microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain. Kinetochore localization was independent of self-association, and driving monomeric CLASP2 to kinetochores fully rescued normal kinetochore–microtubule dynamics, while partially sustaining mitosis. CLASP2 kinetochore localization, recognition of growing microtubule plus-ends through EB–protein interaction, and the ability to associate with curved microtubule protofilaments through TOG2 and TOG3 domains independently sustained normal spindle length, timely spindle assembly checkpoint satisfaction, chromos...
We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of co... more We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as t...
PEX1 and PEX6 are two members of the ATPases Associated with diverse cellular Activities (AAA) fa... more PEX1 and PEX6 are two members of the ATPases Associated with diverse cellular Activities (AAA) family and the core components of the receptor export module (REM) of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex which unfolds substrates by processive threading. However, whether the natural substrate of the PEX1.PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free in vitro system coupled with photoaffinity crosslinking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through it...
Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell... more Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized (35)S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis. To study transport of proteins into peroxisomes, it is possible to use organelle-resident proteins that contain a peroxisomal targeting signal (PTS) as reporters in the assay. In addition, a receptor (PEX5L/S or PEX5L.PEX7) can be used to report the dynamics of shuttling proteins that mediate the import process. Thus, different but complementary perspectives on the mechanism of this pathway can be ...
In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to t... more In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications.
Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 th... more Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interact...
Post-translational modifiers such as the Small Ubiquitin-like Modifier (SUMO) peptide act as fast... more Post-translational modifiers such as the Small Ubiquitin-like Modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has uncovered the key regulatory role that SUMO plays in plant development. Unlike SUMO conjugation pathway components, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. The present study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, uncovering the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an understudied subgroup, Arabidopsis SPF1 and SPF2 were subsequently subjected to functional characterization. Here, loss-of-function mutants implicated both proteins with vegetative growth, flowering time and seed size and yield. Mutants constitutively accumulated SUMO-conjugates, and yeast complementation assays assoc...
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Papers by Jorge Azevedo