A small molecule-directed approach to control protein localization and function

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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Disparate Subcellular Localization Patterns of Pseudomonas aeruginosa Type IV Pilus ATPases Involved in Twitching Motility

Journal of Bacteriology ◽

10.1128/jb.187.3.829-839.2005 ◽

2005 ◽

Vol 187 (3) ◽

pp. 829-839 ◽

Cited By ~ 87

Author(s):

Poney Chiang ◽

Marc Habash ◽

Lori L. Burrows

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescent Protein ◽

Protein Localization ◽

Yellow Fluorescent Protein ◽

Distribution Patterns ◽

Opportunistic Pathogen ◽

Twitching Motility ◽

Polar Localization ◽

Type Iv ◽

And Function

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

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Stage-specific Localization and Expression of c-kit in the Adult Human Testis

Journal of Histochemistry & Cytochemistry ◽

10.1369/jhc.2009.953737 ◽

2009 ◽

Vol 57 (9) ◽

pp. 861-869 ◽

Cited By ~ 41

Author(s):

Sreepoorna K. Unni ◽

Deepak N. Modi ◽

Shilpa G. Pathak ◽

Jayesh V. Dhabalia ◽

Deepa Bhartiya

Keyword(s):

Current Knowledge ◽

Protein Localization ◽

Immunohistochemical Analysis ◽

Human Testis ◽

Specific Expression ◽

Adult Human ◽

Kit Receptor ◽

Specific Localization ◽

Human Spermatogenesis ◽

And Function

The c-kit receptor (KIT) and its ligand, stem cell factor (SCF), represent one of the key regulators of testicular formation, development, and function and have been extensively studied in various animal models. The present study was undertaken to characterize the pattern of localization and expression of c-kit in normal adult human testis. Immunohistochemical analysis showed that KIT is expressed in the cytoplasm of spermatogonia, acrosomal granules of spermatids, and Leydig cells. Interestingly, a rather heterogenous pattern of expression of the protein along the basement membrane was observed. Intense protein localization in spermatogonia was detected in stages I–III, whereas low expression was observed in stages IV–VI of the seminiferous epithelium, indicating that the expression of the molecule was stage specific. In situ hybridization studies revealed that the transcripts of the gene were also localized in a similar non-uniform pattern. To the best of our knowledge, such a stage-specific expression of KIT has not been reported previously in the human testis. The results of the present study may expand current knowledge about the c-kit/SCF system in human spermatogenesis.

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VACCINIA VIRUS COMPLEMENT CONTROL PROTEIN (VCP) IMPROVES KIDNEY STRUCTURE AND FUNCTION FOLLOWING ISCHEMIA/REPERFUSION INJURY IN RATS

Transplantation Journal ◽

10.1097/01.tp.0000331002.33106.57 ◽

2008 ◽

Vol 86 (Supplement) ◽

pp. 601

Author(s):

D Kahn ◽

Y T. Ghebremariam ◽

G Engelbrecht ◽

M Tyler ◽

Z Lotz ◽

...

Keyword(s):

Vaccinia Virus ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Structure And Function ◽

Kidney Structure ◽

Complement Control Protein ◽

And Function ◽

Control Protein

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Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET

Biophysical Journal ◽

10.1016/j.bpj.2016.11.1297 ◽

2017 ◽

Vol 112 (3) ◽

pp. 237a

Author(s):

Piyali Guhathakurta ◽

Ewa Prochniewicz ◽

Kurt C. Peterson ◽

Benjamin D. Grant ◽

Gregory D. Gillispie ◽

...

Keyword(s):

Small Molecule ◽

High Throughput ◽

Structure And Function ◽

Throughput Time ◽

Time Resolved ◽

Small Molecule Modulators ◽

And Function

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Determinants of Drosophila zw10 protein localization and function

Journal of Cell Science ◽

10.1242/jcs.107.4.785 ◽

1994 ◽

Vol 107 (4) ◽

pp. 785-798 ◽

Cited By ~ 13

Author(s):

B.C. Williams ◽

M.L. Goldberg

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Protein Localization ◽

Metaphase Plate ◽

Obvious Effect ◽

Anaphase Onset ◽

Protein Functions ◽

Kinetochore Region ◽

And Function ◽

Feedback Pathway

We have examined several issues concerning how the Drosophila l(1)zw10 gene product functions to ensure proper chromosome segregation. (a) We have found that in zw10 mutant embryos and larval neuroblasts, absence of the zw10 protein has no obvious effect on either the congression of chromosomes to the metaphase plate or the morphology of the metaphase spindle, although many aberrations are observed subsequently in anaphase. This suggests that activity of the zw10 protein becomes essential at anaphase onset, a time at which the zw10 protein is redistributed to the kinetochore region of the chromosomes. (b) The zw10 protein appears to bind to kinetochores in mitotically arrested cells, eventually accumulating to high levels within the chromosome mass. Our results imply that zw10 may act as part of a novel feedback pathway that normally renders sister chromatid separation dependent upon spindle integrity. (c) The localization of zw10 protein is altered by two mitotic mutations, rough deal and abnormal anaphase resolution, that specifically disrupt anaphase. These findings indicate that the zw10 protein functions as part of a multicomponent mechanism ensuring proper chromosome segregation at the beginning of anaphase.

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Hypoxia elicits broad and systematic changes in protein subcellular localization

AJP Cell Physiology ◽

10.1152/ajpcell.00481.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. C913-C928 ◽

Cited By ~ 17

Author(s):

Robert Michael Henke ◽

Ranita Ghosh Dastidar ◽

Ajit Shah ◽

Daniela Cadinu ◽

Xiao Yao ◽

...

Keyword(s):

Protein Function ◽

Protein Localization ◽

Cell Periphery ◽

Protein Distribution ◽

Yeast Saccharomyces Cerevisiae ◽

Transcriptional Regulatory ◽

Chromatin Remodeling Complexes ◽

Oxygen Signaling ◽

Cellular Compartments ◽

Control Protein

Oxygen provides a crucial energy source in eukaryotic cells. Hence, eukaryotes ranging from yeast to humans have developed sophisticated mechanisms to respond to changes in oxygen levels. Regulation of protein localization, like protein modifications, can be an effective mechanism to control protein function and activity. However, the contribution of protein localization in oxygen signaling has not been examined on a genomewide scale. Here, we examine how hypoxia affects protein distribution on a genomewide scale in the model eukaryote, the yeast Saccharomyces cerevisiae . We demonstrate, by live cell imaging, that hypoxia alters the cellular distribution of 203 proteins in yeast. These hypoxia-redistributed proteins include an array of proteins with important functions in various organelles. Many of them are nuclear and are components of key regulatory complexes, such as transcriptional regulatory and chromatin remodeling complexes. Under hypoxia, these proteins are synthesized and retained in the cytosol. Upon reoxygenation, they relocalize effectively to their normal cellular compartments, such as the nucleus, mitochondria, endoplasmic reticulum, and cell periphery. The resumption of the normal cellular locations of many proteins can occur even when protein synthesis is inhibited. Furthermore, we show that the changes in protein distribution induced by hypoxia follow a slower trajectory than those induced by reoxygenation. These results show that the regulation of protein localization is a common and potentially dominant mechanism underlying oxygen signaling and regulation. These results may have broad implications in understanding oxygen signaling and hypoxia responses in higher eukaryotes such as humans.

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LIM kinase1 regulates mitotic centrosome integrity via its activity on dynein light intermediate chains

Open Biology ◽

10.1098/rsob.170202 ◽

2018 ◽

Vol 8 (6) ◽

pp. 170202 ◽

Cited By ~ 2

Author(s):

Sirong Ou ◽

Mei-Hua Tan ◽

Ting Weng ◽

HoiYeung Li ◽

Cheng-Gee Koh

Keyword(s):

Protein Transport ◽

Protein Localization ◽

Cytoplasmic Dynein ◽

Spindle Pole ◽

Centrosomal Protein ◽

And Function ◽

Cytoskeleton Dynamics ◽

Dynein Light Intermediate Chains ◽

Intermediate Chains ◽

Mitotic Spindle Pole

Abnormal centrosome number and function have been implicated in tumour development. LIM kinase1 (LIMK1), a regulator of actin cytoskeleton dynamics, is found to localize at the mitotic centrosome. However, its role at the centrosome is not fully explored. Here, we report that LIMK1 depletion resulted in multi-polar spindles and defocusing of centrosomes, implicating its involvement in the regulation of mitotic centrosome integrity. LIMK1 could influence centrosome integrity by modulating centrosomal protein localization at the spindle pole. Interestingly, dynein light intermediate chains (LICs) are able to rescue the defects observed in LIMK1-depleted cells. We found that LICs are potential novel interacting partners and substrates of LIMK1 and that LIMK1 phosphorylation regulates cytoplasmic dynein function in centrosomal protein transport, which in turn impacts mitotic spindle pole integrity.

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