Dataset Information

Phosphorylation of multiple sites within an acidic region of Alcadein ? is required for kinesin-1 association and Golgi exit of Alcadein ? cargo.

ABSTRACT: Alcadein ? (Alc?) is a major cargo of kinesin-1 that is subjected to anterograde transport in neuronal axons. Two tryptophan- and aspartic acid-containing (WD) motifs located in its cytoplasmic domain directly bind the tetratricopeptide repeat (TPR) motifs of the kinesin light chain (KLC), which activate kinesin-1 and recruit kinesin-1 to Alc? cargo. We found that phosphorylation of three serine residues in the acidic region located between the two WD motifs is required for interaction with KLC. Phosphorylation of these serine residues may alter the disordered structure of the acidic region to induce direct association with KLC. Replacement of these serines with Ala results in a mutant that is unable to bind kinesin-1, which impairs exit of Alc? cargo from the Golgi. Despite this deficiency, the compromised Alc? mutant was still transported, albeit improperly by vesicles following missorting of the Alc? mutant with amyloid ?-protein precursor (APP) cargo. This suggests that APP partially compensates for defective Alc? in anterograde transport by providing an alternative cargo receptor for kinesin-1.

SUBMITTER: Sobu Y

PROVIDER: S-EPMC5739299 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo.

Sobu Yuriko Y Furukori Keiko K Chiba Kyoko K Nairn Angus C AC Kinjo Masataka M Hata Saori S Suzuki Toshiharu T

Molecular biology of the cell 20171101 26

Alcadein α (Alcα) is a major cargo of kinesin-1 that is subjected to anterograde transport in neuronal axons. Two tryptophan- and aspartic acid-containing (WD) motifs located in its cytoplasmic domain directly bind the tetratricopeptide repeat (TPR) motifs of the kinesin light chain (KLC), which activate kinesin-1 and recruit kinesin-1 to Alcα cargo. We found that phosphorylation of three serine residues in the acidic region located between the two WD motifs is required for interaction with KLC. ...[more]

PMID: 29093024

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Project description: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.

Dataset Information

Phosphorylation of multiple sites within an acidic region of Alcadein ? is required for kinesin-1 association and Golgi exit of Alcadein ? cargo.

Publications

Phosphorylation of multiple sites within an acidic region of Alcadein α is required for kinesin-1 association and Golgi exit of Alcadein α cargo.

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