scholarly journals The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization

2020 ◽  
Vol 133 (16) ◽  
pp. jcs245415
Author(s):  
Arpita Sen ◽  
Wen-Chieh Hsieh ◽  
Claudia B. Hanna ◽  
Chuan-Chih Hsu ◽  
McKeith Pearson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Turnover ◽  
Spatial Organization ◽  
Protein Sorting ◽  
Cytoplasmic Tail ◽  
Phosphorylation Sites ◽  
Na Pump ◽  
Substantial Progress ◽  
Casein Kinases ◽  
Standing Question

ABSTRACTIt is well known that in addition to its classical role in protein turnover, ubiquitylation is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K1090, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.

scholarly journals Ubiquitination and Phosphorylation are Independently Required for Epsin-Mediated Internalization of Cargo in S. cerevisiae

2020 ◽  
Author(s):  
Arpita Sen ◽  
Wen-Chieh Hsieh ◽  
Claudia B. Hanna ◽  
Chuan-Chih Hsu ◽  
McKeith Pearson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein Turnover ◽  
Coincidence Detection ◽  
Dependent Manner ◽  
Detection Mechanism ◽  
Knock Out ◽  
Substantial Progress ◽  
Casein Kinases ◽  
Dual Detection ◽  
Standing Question

ABSTRACTIt is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains: given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes?Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na+ pump Ena1.Internalization of Ena1-GFP was abolished in double epsin knock-out in S. cerevisiae and was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub Interacting Motif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys1090 is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3.We determined that in addition to ubiquitination of K1090, the presence of a Ser/Thr-rich patch (S1076TST1079) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K1090 and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1.We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.

Protein Sorting in Healthy and Diseased Photoreceptors

2019 ◽  
Vol 5 (1) ◽  
pp. 73-98 ◽  
Author(s):  
Yoshikazu Imanishi
Keyword(s):  
Signal Transduction ◽  
Membrane Protein ◽  
Outer Segment ◽  
Protein Sorting ◽  
Rods And Cones ◽  
Retinal Photoreceptor ◽  
Photoreceptor Neurons ◽  
Cellular Compartments ◽  
G Protein Coupled ◽  
Distinct Membrane

Rods and cones are retinal photoreceptor neurons required for our visual sensation. Because of their highly polarized structures and well-characterized processes of G protein–coupled receptor–mediated phototransduction signaling, these photoreceptors have been excellent models for studying the compartmentalization and sorting of proteins. Rods and cones have a modified ciliary compartment called the outer segment (OS) as well as non-OS compartments. The distinct membrane protein compositions between OS and non-OS compartments suggest that the OS is separated from the rest of the cellular compartments by multiple barriers or gates that are selectively permissive to specific cargoes. This review discusses the mechanisms of protein sorting and compartmentalization in photoreceptor neurons. Proper sorting and compartmentalization of membrane proteins are required for signal transduction and transmission. This review also discusses the roles of compartmentalized signaling, which is compromised in various retinal ciliopathies.

scholarly journals Akr1p and the Type I Casein Kinases Act prior to the Ubiquitination Step of Yeast Endocytosis: Akr1p Is Required for Kinase Localization to the Plasma Membrane

2000 ◽  
Vol 20 (14) ◽  
pp. 5350-5359 ◽  
Author(s):  
Ying Feng ◽  
Nicholas G. Davis
Keyword(s):  
Plasma Membrane ◽  
Deletion Mutant ◽  
Ankyrin Repeat ◽  
Type I ◽  
Phosphorylation Sites ◽  
Cytoplasmic Localization ◽  
Lipid Modification ◽  
Wild Type ◽  
Ankyrin Repeat Protein ◽  
Casein Kinases

ABSTRACT Ubiquitination of the plasma membrane-localized yeast a-factor receptor (Ste3p) triggers a rapid, ligand-independent endocytosis leading to its vacuolar degradation. This report identifies two mutants that block uptake by blocking ubiquitination, these being mutant either for the ankyrin repeat protein Akr1p or for the redundant type I casein kinases Yck1p and Yck2p. While no obvious defect was seen for wild-type Ste3p phosphorylation in akr1 or yck mutant backgrounds, examination of the Δ320-413 Ste3p deletion mutant phosphorylation did reveal a clear defect in both mutants. The Δ320-413 deletion removes 18 Ser-Thr residues (possible YCK-independent phosphorylation sites) yet retains the 15 Ser-Thr residues of the Ste3p PEST-like ubiquitination-endocytosis signal. Two other phenotypes link akr1 and yck mutants: both are defective in phosphorylation of wild-type α-factor receptor, and while both are defective for Ste3p constitutive internalization, both remain partially competent for the Ste3p ligand-dependent uptake mode. Yck1p-Yck2p may be the function responsible in phosphorylation of the PEST-like ubiquitination-endocytosis signal. Akr1p appears to function in localizing Yck1p-Yck2p to the plasma membrane, a localization that depends on prenylation of C-terminal dicysteinyl motifs. In akr1Δ cells, Yck2p is mislocalized, showing a diffuse cytoplasmic localization identical to that seen for a Yck2p mutant that lacks the C-terminal Cys-Cys, indicating a likely Akr1p requirement for the lipid modification of Yck2p, for prenylation, or possibly for palmitoylation.

Loss of a GPI-Anchored Membrane Protein Aah3p Causes a Defect in Vacuolar Protein Sorting inSchizosaccharomyces pombe

2007 ◽  
Vol 71 (2) ◽  
pp. 623-626 ◽  
Author(s):  
Tomoko IWAKI ◽  
Tomotake MORITA ◽  
Naotaka TANAKA ◽  
Yuko GIGA-HAMA ◽  
Kaoru TAKEGAWA
Keyword(s):  
Membrane Protein ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Vacuolar Protein

scholarly journals Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

1993 ◽  
Vol 292 (3) ◽  
pp. 833-838 ◽  
Author(s):  
O Rosorius ◽  
G Mieskes ◽  
O G Issinger ◽  
C Körner ◽  
B Schmidt ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
Phosphorylation Sites ◽  
Calmodulin Kinase ◽  
Major Species ◽  
Mannose 6 Phosphate ◽  
Inhibition Studies

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.

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