scholarly journals A Rab Requirement Is Not Bypassed in SLY1-20 Suppression

2005 ◽  
Vol 16 (4) ◽  
pp. 1839-1849 ◽  
Author(s):  
Nicole Ballew ◽  
Yiting Liu ◽  
Charles Barlowe
Keyword(s):  
Golgi Complex ◽  
Rab Gtpase ◽  
Loss Of Function ◽  
Biochemical Tests ◽  
Anterograde Transport ◽  
Vesicle Tethering ◽  
Rab Protein ◽  
Complex Membrane ◽  
Conserved Oligomeric Golgi Complex ◽  
Gdp Dissociation Inhibitor

The Rab GTPase Ypt1p and the large homodimer Uso1p are both required for tethering endoplasmic reticulum-derived vesicles to early Golgi compartments in yeast. Loss-of-function ypt1 and uso1 mutations are suppressed by SLY1-20, a dominant allele that encodes the Sed5p-associated protein, Sly1p. Here, we investigate the mechanism of SLY1-20 suppression. In wild-type strains, Ypt1p can be coimmunoprecipitated with Uso1p; however, in a ypt1Δ/SLY1-20 strain, which lacks this complex, membrane binding of Uso1p was reduced. In spite of Ypt1p depletion, Uso1p-dependent vesicle tethering was not bypassed under the ypt1Δ/SLY1-20 condition. Moreover, tethering and fusion assays with ypt1Δ/SLY1-20 membranes remained sensitive to Rab GDP dissociation inhibitor. These results indicate that an alternative Rab protein satisfies the Ypt1p requirement in Uso1p-dependent tethering when SLY1-20 is expressed. Further genetic and biochemical tests revealed that a related Rab protein, Ypt6, might substitute for Ypt1p in ypt1Δ/SLY1-20 cells. Additional experimentation to address the mechanism of SLY1-20 suppression in a cog2Δ [sec35Δ] strain indicated that the Cog2p subunit of the conserved oligomeric Golgi complex is either functionally redundant or is not directly required for anterograde transport to the Golgi complex.

scholarly journals The ER v-SNAREs are required for GPI-anchored protein sorting from other secretory proteins upon exit from the ER

2003 ◽  
Vol 162 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Pierre Morsomme ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman
Keyword(s):  
Golgi Complex ◽  
Protein Sorting ◽  
Secretory Proteins ◽  
Rab Gtpase ◽  
Cargo Protein ◽  
Conserved Oligomeric Golgi Complex ◽  
Sorting Process ◽  
Conserved Oligomeric Golgi

Glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in distinct vesicles from other secretory proteins, and this sorting event requires the Rab GTPase Ypt1p, tethering factors Uso1p, and the conserved oligomeric Golgi complex. Here we show that proper sorting depended on the vSNAREs, Bos1p, Bet1p, and Sec22p. However, the t-SNARE Sed5p was not required for protein sorting upon ER exit. Moreover, the sorting defect observed in vitro with bos1–1 extracts was also observed in vivo and was visualized by EM. Finally, transport and maturation of the GPI-anchored protein Gas1p was specifically affected in a bos1–1 mutant at semirestrictive temperature. Therefore, we propose that v-SNAREs are part of the cargo protein sorting machinery upon exit from the ER and that a correct sorting process is necessary for proper maturation of GPI-anchored proteins.

scholarly journals Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

1999 ◽  
Vol 147 (4) ◽  
pp. 729-742 ◽  
Author(s):  
Susan M. VanRheenen ◽  
Xiaochun Cao ◽  
Stephanie K. Sapperstein ◽  
Elbert C. Chiang ◽  
Vladimir V. Lupashin ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Rab Gtpase ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dominant Form ◽  
Vesicle Tethering ◽  
Protein Receptor ◽  
Complex Target

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well.

LRRK2 and Rab GTPases

2018 ◽  
Vol 46 (6) ◽  
pp. 1707-1712 ◽  
Author(s):  
Suzanne R. Pfeffer
Keyword(s):  
Membrane Trafficking ◽  
Protein Function ◽  
Short Review ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Complex Interactions ◽  
Preferential Binding ◽  
Bona Fide ◽  
Mutant Lrrk2

Leucine-rich repeat kinase 2 (LRRK2) is mutated in familial Parkinson's disease, and pathogenic mutations activate the kinase activity. A tour de force screen by Mann and Alessi and co-workers identified a subset of Rab GTPases as bona fide LRRK2 substrates. Rab GTPases are master regulators of membrane trafficking and this short review will summarize what we know about the connection between LRRK2 and this family of regulatory proteins. While, in most cases, Rab GTPase phosphorylation is predicted to interfere with Rab protein function, the discovery of proteins that show preferential binding to phosphorylated Rabs suggests that more complex interactions may also contribute to mutant LRRK2-mediated pathology.

Biochemical investigation of rheumatic diseases

2013 ◽  
pp. 451-456
Author(s):  
Berenice Lopez ◽  
Patrick J. Twomey
Keyword(s):  
Rheumatic Diseases ◽  
Wide Spectrum ◽  
Imaging Techniques ◽  
Measurement Technology ◽  
Loss Of Function ◽  
Sample Collection ◽  
Biochemical Tests ◽  
Metabolic Bone ◽  
Routine Monitoring ◽  
Organ Specific

It is important for rheumatologists to have an understanding of biochemical tests including an awareness of their limitations. The biological variability of an analyte both within and between individuals, the limitations of the measurement technology, the sensitivity of laboratory internal quality control and external quality assurance procedures, as well as interlaboratory variations in practices including sample collection procedures, may all impact on the interpretation of a result. Biochemical tests are often requested to monitor organ-specific dysfunction arising as an adverse consequence of pharmacotherapy or as a component of a systemic rheumatic disease, although dysfunction may also reflect infection or coincidental pathology. Patients with rheumatic diseases are at high risk of renal and hepatic disease. Serum creatinine and its derivative estimated glomerular filtration rate (eGFR) are the most readily available surrogate markers of GFR and are used to assess renal impairment and monitor its course. However, the use of creatinine alone lacks sensitivity and a substantial loss of function must occur before creatinine levels are increased. Additional biochemical screening for kidney damage can be performed by assessment of glomerular integrity, including proteinuria or albuminuria and haematuria. A wide spectrum of rheumatic diseases can affect the liver with various degrees of involvement and hepatic pathology. These often present with cholestatic or hepatitic biochemical profiles. The medical management of rheumatic diseases also involves medications that are hepatotoxic, and routine monitoring of liver function is recommended. This approach is not problem-free and may be improved by quantitative determinations of non-invasive markers of liver fibrosis in the future. Together with imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease.

scholarly journals Conserved oligomeric Golgi complex specifically regulates the maintenance of Golgi glycosylation machinery

Glycobiology ◽  
2011 ◽  
Vol 21 (12) ◽  
pp. 1554-1569 ◽  
Author(s):  
Irina D Pokrovskaya ◽  
Rose Willett ◽  
Richard D Smith ◽  
Willy Morelle ◽  
Tetyana Kudlyk ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Conserved Oligomeric Golgi Complex ◽  
Conserved Oligomeric Golgi

Endoplasmic reticulum–Golgi complex membrane contact sites

2015 ◽  
Vol 35 ◽  
pp. 43-50 ◽  
Author(s):  
Maria Antonietta De Matteis ◽  
Laura Rita Rega
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Complex Membrane ◽  
Membrane Contact

scholarly journals Rab6 is required for rapid, cisternal-specific, intra-Golgi cargo transport

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lindsey James Dickson ◽  
Shijie Liu ◽  
Brian Storrie
Keyword(s):  
Super Resolution ◽  
Small Gtpase ◽  
Loss Of Function ◽  
Anterograde Transport ◽  
Surface Appearance ◽  
Progression Model ◽  
Cargo Transport ◽  
Marker Proteins ◽  
Cargo Protein ◽  
Biochemical Approach

Abstract Rab6, the most abundant Golgi associated small GTPase, consists of 2 equally common isoforms, Rab6A and Rab6A′, that differ in 3 amino acids and localize to trans Golgi cisternae. The two isoforms are largely redundant in function and hence are often referred to generically as Rab6. Rab6 loss-of-function inhibits retrograde Golgi trafficking, induces an increase in Golgi cisternal number in HeLa cells and delays the cell surface appearance of the anterograde cargo protein, VSVG. We hypothesized that these effects are linked and might be explained by a cisternal-specific delay in cargo transport. In pulse chase experiments using a deconvolved, confocal line scanning approach to score the distribution of the tsO45 mutant of VSVG protein in Rab6 depleted cells, we found that anterograde transport at 32 °C, permissive conditions, through the Golgi apparatus was locally delayed, almost tenfold, between medial and trans Golgi cisterna. Cis to medial transport was nearly normal as was trans Golgi to TGN transport. TGN exit was unaffected by Rab6 depletion. These effects were the same with either of two siRNAs. Similar intra-Golgi transport delays were seen at 37 °C with RUSH VSVG or a RUSH GPI-anchored construct using a biotin pulse to release the marker proteins from the ER. Using 3D-SIM, a super resolution approach, we found that RUSH VSVG transport was delayed pre-trans Golgi. These visual approaches suggest a selective slowing of anterograde transport relative to 3 different marker proteins downstream of the trans Golgi. Using a biochemical approach, we found that the onset of VSVG endoglycosidase H resistance in Rab6 depleted cells was delayed. Depletion of neither Rab6A or Rab6A′ isoforms alone had any effect on anterograde transport through the Golgi suggesting that Rab6A and Rab6A′ act coordinately. Delayed cargo transport conditions correlate strongly with a proliferation of Golgi cisternae observed in earlier electron microscopy. Our results strongly indicate that Rab6 is selectively required for rapid anterograde transport from the medial to trans Golgi. We suggest that the observed correlation with localized cisternal proliferation fits best with a cisternal progression model of Golgi function.

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