Regulation of Golgi structure and function by ARF-like protein 1 (Arl1)

2001 ◽  
Vol 114 (24) ◽  
pp. 4543-4555 ◽  
Author(s):  
Lei Lu ◽  
Heinz Horstmann ◽  
Cheepeng Ng ◽  
Wanjin Hong
Keyword(s):  
Golgi Apparatus ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Structure And Function ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intact Cells ◽  
Cellular Events ◽  
Golgi Structure ◽  
And Function

Arl1 is a member of the ARF-like protein (Arl) subfamily of small GTPases. Nothing is known about the function of Arl1 except for the fact that it is essential for normal development in Drosophila and that it is associated with the Golgi apparatus. In this study, we first demonstrate that Arl1 is enriched at the trans side of the Golgi, marked by AP-1. Association of Arl1 with the Golgi is saturable in intact cells and depends on N-terminal myristoylation. Over-expression of Arl1(T31N), which is expected to be restricted to the GDP-bound form and thus function as a dominant-negative mutant, causes the disappearance of the Golgi apparatus (marked by Golgi SNARE GS28), suggesting that Arl1 is necessary for maintaining normal Golgi structure. Overexpression of Arl1(Q71L), a mutant restricted primarily to the activated GTP-bound form, causes an expansion of the Golgi apparatus with massive and stable Golgi association of COPI and AP-1 coats. Interestingly, Golgi ARFs also become stably associated with the expanded Golgi. Transport of the envelope protein of vesicular stomatitis virus (VSV-G) along the secretory pathway is arrested at the expanded Golgi upon expression of Arl1(Q71L). The structure of stacked cisternae of the Golgi is disrupted in cells expressing Arl1(Q71L), resulting in the transformation of the Golgi into an extensive vesicule-tubule network. In addition, the GTP form of Arl1 interacts with arfaptin-2/POR1 but not GGA1, both of which interact with GTP-restricted ARF1, suggesting that Arl1 and ARF1 share some common effectors in regulating cellular events. On the basis of these observations, we propose that one of the mechanisms for the cell to regulate the structure and function of the Golgi apparatus is through the action of Arl1.

TBC proteins: GAPs for mammalian small GTPase Rab?

2011 ◽  
Vol 31 (3) ◽  
pp. 159-168 ◽  
Author(s):  
Mitsunori Fukuda
Keyword(s):  
Insulin Resistance ◽  
Small Gtpases ◽  
Structure And Function ◽  
Small Gtpase ◽  
Cell Cycle Regulators ◽  
Gtpase Activating Protein ◽  
Domain Family ◽  
Protein Motif ◽  
Conserved Domain ◽  
And Function

The TBC (Tre-2/Bub2/Cdc16) domain was originally identified as a conserved domain among the tre-2 oncogene product and the yeast cell cycle regulators Bub2 and Cdc16, and it is now widely recognized as a conserved protein motif that consists of approx. 200 amino acids in all eukaryotes. Since the TBC domain of yeast Gyps [GAP (GTPase-activating protein) for Ypt proteins] has been shown to function as a GAP domain for small GTPase Ypt/Rab, TBC domain-containing proteins (TBC proteins) in other species are also expected to function as a certain Rab-GAP. More than 40 different TBC proteins are present in humans and mice, and recent accumulating evidence has indicated that certain mammalian TBC proteins actually function as a specific Rab-GAP. Some mammalian TBC proteins {e.g. TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1] and TBC1D4/AS160 (Akt substrate of 160 kDa)} play an important role in homoeostasis in mammals, and defects in them are directly associated with mouse and human diseases (e.g. leanness in mice and insulin resistance in humans). The present study reviews the structure and function of mammalian TBC proteins, especially in relation to Rab small GTPases.

scholarly journals Calculating glycoprotein similarities from mass spectrometric data

2020 ◽  
pp. mcp.R120.002223
Author(s):  
William Edwin Hackett ◽  
Joseph Zaia
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Biological Effects ◽  
Life Forms ◽  
Structure And Function ◽  
Mass Spectrometric ◽  
Protein Quantification ◽  
Protein Glycosylation ◽  
Spectrometric Data ◽  
And Function

Complex protein glycosylation occurs through biosynthetic steps in the secretory pathway that create macro- and microheterogeneity of structure and function.  Required for all life forms, glycosylation diversifies and adapts protein interactions with binding partners that underpin interactions at cell surfaces and pericellular and extracellular environments. Because these biological effects arise from heterogeneity of structure and function, it is necessary to measure their changes as part of the quest to understand nature.  Quite often, however, the assumption behind proteomics that post-translational modifications are discrete additions that can be modeled using the genome as a template does not apply to protein glycosylation.  Rather, it is necessary to quantify the glycosylation distribution at each glycosite and to aggregate this information into a population of mature glycoproteins that exist in a given biological system.  To date, mass spectrometric methods for assigning singly glycosylated peptides are well-established.  But it is necessary to quantify glycosylation heterogeneity accurately in order to gauge the alterations that occur during biological processes.  The task is to quantify the glycosylated peptide forms as accurately as possible and then apply appropriate bioinformatics algorithms to the calculation of micro- and macro-similarities.  In this review, we summarize current approaches for protein quantification as they apply to this glycoprotein similarity problem.

Illuminating cellular structure and function in the early secretory pathway by multispectral 3D imaging in living cells

2000 ◽  
Author(s):  
Jens Rietdorf ◽  
David J. Stephens ◽  
Anthony Squire ◽  
Jeremy Simpson ◽  
David T. Shima ◽  
...  
Keyword(s):  
Cellular Structure ◽  
3D Imaging ◽  
Secretory Pathway ◽  
Living Cells ◽  
Structure And Function ◽  
Early Secretory Pathway ◽  
And Function

Properties and metabolism of the aqueous cytoplasm and its boundaries

1984 ◽  
Vol 246 (2) ◽  
pp. R133-R151 ◽  
Author(s):  
J. S. Clegg
Keyword(s):  
Cell Biology ◽  
Cell Structure ◽  
Large Fraction ◽  
Pure Water ◽  
Cell Ultrastructure ◽  
Structure And Function ◽  
Cell Water ◽  
Animal Cells ◽  
Intact Cells ◽  
And Function

The nucleoplasm, the interiors of cytoplasmic membrane-bound organelles, and the aqueous cytoplasm make up the aqueous compartments of animal cells. The extent to which these compartments are concentrated solutions of macromolecules, metabolites, ions, and other solutes is a matter of some importance to current thinking about cell structure and function. This paper will focus on the aqueous cytoplasm. It will show that the composition and metabolic activities of the cytosol, obtained by methods of cell disruption and fractionation, bear almost no resemblance to those of the aqueous cytoplasm in intact cells. The consequences of this to contemporary views on cell structure and function are considered. A closely related topic concerns the physical properties of the dominant component of these compartments, water: Are these properties the same as those of water in aqueous solutions, or are they altered as a result of interaction with cell architecture? Available evidence strongly suggests that at least a large fraction of the total cell water exhibits properties that markedly differ from those of pure water. Selected examples of these studies will be reviewed, and the roles of cell water will be discussed, notably as they relate to metabolism and cell ultrastructure. Although dimly perceived at present, it appears that living cells exhibit an organization far greater than the current teachings of cell biology reveal.

scholarly journals Intracellular water and the cytomatrix: some methods of study and current views.

1984 ◽  
Vol 99 (1) ◽  
pp. 167s-171s ◽  
Author(s):  
J S Clegg
Keyword(s):  
Cell Biology ◽  
Cellular Structure ◽  
Large Fraction ◽  
Cell Ultrastructure ◽  
Structure And Function ◽  
Intracellular Water ◽  
Cell Fractionation ◽  
Intact Cells ◽  
Dilute Aqueous Solutions ◽  
And Function

The extent to which the properties of water in cells are like those of water in dilute aqueous solutions is a question of broad significance to cell biology. A detailed answer is not available at present, although evidence is accumulating that the properties of at least a large fraction of intracellular water are altered by interactions with cell ultrastructure, notably the cytomatrix. That and related evidence also suggests that the properties, composition, and activities of the "aqueous cytoplasm" of intact cells bear little resemblance to those of the "cytosol" obtained by cell fractionation. This paper will consider some of the evidence for these possibilities and some of their potential consequences with regard to cellular structure and function.

scholarly journals Exploring the Structure and Function of the Mycobacterial KatG Protein Using trans-Dominant Mutants

2003 ◽  
Vol 47 (1) ◽  
pp. 188-195 ◽  
Author(s):  
Joseph A. DeVito ◽  
Sheldon Morris
Keyword(s):  
Structure And Function ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Active Site Residues ◽  
Site Mutation ◽  
Dominant Negative Mutation ◽  
Cell Extracts ◽  
Catalase Peroxidase ◽  
And Function ◽  
Dominant Mutants

ABSTRACT In order to probe the structure and function of the mycobacterial catalase-peroxidase enzyme (KatG), we employed a genetic approach using dominant-negative analysis of katG merodiploids. Transformation of Mycobacterium bovis BCG with various katG point mutants (expressed from low-copy-number plasmids) resulted in reductions in peroxidase and catalase activities as measured in cell extracts. These reductions in enzymatic activity usually correlated with increased resistance to the antituberculosis drug isoniazid (INH). However, for the N138S trans-dominant mutant, the catalase-peroxidase activity was significantly decreased while the sensitivity to INH was retained. trans-dominance required katG expression from multicopy plasmids and could not be demonstrated with katG mutants integrated elsewhere on the wild-type M. bovis BCG chromosome. Reversal of the mutant phenotype through plasmid exchange suggested the catalase-peroxidase deficiency occurred at the protein level and that INH resistance was not due to a second site mutation(s). Electrophoretic analysis of KatG proteins from the trans-dominant mutants showed a reduction in KatG dimers compared to WT and formation of heterodimers with reduced activity. The mutants responsible for these defects cluster around proposed active site residues: N138S, T275P, S315T, and D381G. In an attempt to identify mutants that might delimit the region(s) of KatG involved in subunit interactions, C-terminal truncations were constructed (with and without the D381G dominant-negative mutation). None of the C-terminal deletions were able to complement a ΔkatG strain, nor could they cause a dominant-negative effect on the WT. Taken together, these results suggest an intricate association between the amino- and carboxy-terminal regions of KatG and may be consistent with a domain-swapping mechanism for KatG dimer formation.

scholarly journals Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

1998 ◽  
Vol 9 (10) ◽  
pp. 2819-2837 ◽  
Author(s):  
Sara Jones ◽  
Celeste J. Richardson ◽  
Robert J. Litt ◽  
Nava Segev
Keyword(s):  
Endoplasmic Reticulum ◽  
Secretory Pathway ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Rab Proteins ◽  
Nucleotide Exchange ◽  
Gtp Hydrolysis ◽  
Golgi Transport ◽  
Transport Assay

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7and GYP1, nor is it influenced by mutations inSEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

scholarly journals Fundamental Characteristics of AAA+ Protein Family Structure and Function

Archaea ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Justin M. Miller ◽  
Eric J. Enemark
Keyword(s):  
Atp Hydrolysis ◽  
Molecular Machines ◽  
Ring Structure ◽  
Structure And Function ◽  
Central Channel ◽  
Aaa Atpase ◽  
Cellular Events ◽  
Critical Components ◽  
And Function ◽  
Aaa Protein

Many complex cellular events depend on multiprotein complexes known as molecular machines to efficiently couple the energy derived from adenosine triphosphate hydrolysis to the generation of mechanical force. Members of the AAA+ ATPase superfamily (ATPases Associated with various cellular Activities) are critical components of many molecular machines. AAA+ proteins are defined by conserved modules that precisely position the active site elements of two adjacent subunits to catalyze ATP hydrolysis. In many cases, AAA+ proteins form a ring structure that translocates a polymeric substrate through the central channel using specialized loops that project into the central channel. We discuss the major features of AAA+ protein structure and function with an emphasis on pivotal aspects elucidated with archaeal proteins.

scholarly journals Binding of Herpes Simplex Virus 1 UL20 to GODZ (DHHC3) Affects Its Palmitoylation and Is Essential for Infectivity and Proper Targeting and Localization of UL20 and Glycoprotein K

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Shaohui Wang ◽  
Kevin R. Mott ◽  
Kolja Wawrowsky ◽  
Konstantin G. Kousoulas ◽  
Bernhard Luscher ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Envelope Protein ◽  
Zinc Finger Protein ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Herpes Simplex Virus 1 ◽  
Finger Protein ◽  
Glycoprotein K ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) UL20 plays a crucial role in the envelopment of the cytoplasmic virion and its egress. It is a nonglycosylated envelope protein that is regulated as a γ1 gene. Two-hybrid and pulldown assays demonstrated that UL20, but no other HSV-1 gene-encoded proteins, binds specifically to GODZ (also known as DHHC3), a cellular Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein. A catalytically inactive dominant-negative GODZ construct significantly reduced HSV-1 replication in vitro and affected the localization of UL20 and glycoprotein K (gK) and their interactions but not glycoprotein C (gC). GODZ is involved in palmitoylation, and we found that UL20 is palmitoylated by GODZ using a GODZ dominant-negative plasmid. Blocking of palmitoylation using 2-bromopalmitate (2-BP) affected the virus titer and the interaction of UL20 and gK but did not affect the levels of these proteins. In conclusion, we have shown that binding of UL20 to GODZ in the Golgi apparatus regulates trafficking of UL20 and its subsequent effects on gK localization and virus replication. We also have demonstrated that GODZ-mediated UL20 palmitoylation is critical for UL20 membrane targeting and thus gK cell surface expression, providing new mechanistic insights into how UL20 palmitoylation regulates HSV-1 infectivity. IMPORTANCE HSV-1 UL20 is a nonglycosylated essential envelope protein that is highly conserved among herpesviruses. In this study, we show that (i) HSV-1 UL20 binds to GODZ (also known as DHHC3), a Golgi apparatus-specific Asp-His-His-Cys (DHHC) zinc finger protein; (ii) a GODZ dominant-negative mutant and an inhibitor of palmitoylation reduced HSV-1 titers and altered the localization of UL20 and glycoprotein K; and (iii) UL20 is palmitoylated by GODZ, and this UL20 palmitoylation is required for HSV-1 infectivity. Thus, blocking of the interaction of UL20 with GODZ, using a GODZ dominant-negative mutant or possibly GODZ shRNA, should be considered a potential alternative therapy in not only HSV-1 but also other conditions in which GODZ processing is an integral component of pathogenesis.

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