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. 2020 Aug 26;53(4):93-97.
doi: 10.1267/ahc.20-00006. Epub 2020 Jul 22.

Rab35 Targeting to the Plasma Membrane Is Dependent on the C-terminal Polybasic Cluster

Katsuhisa Kawai  1 , Youhei Egami  1 , Arata Nishigaki  1 , Nobukazu Araki  1
Affiliations

Rab35 Targeting to the Plasma Membrane Is Dependent on the C-terminal Polybasic Cluster

Katsuhisa Kawai et al. Acta Histochem Cytochem. .

Abstract

Rab35, a member of the Rab GTPase family, has been implicated in various cellular processes including cell motility and membrane trafficking. Although Rab35 is localized to the plasma membrane, Rab proteins that are identified to have high sequence homology with Rab35 exhibit distinct subcellular localization patterns. Comparing the amino acid sequences between Rab35 and its family members revealed a significant variation in an approximate 30-amino acid region of the C-terminus. This suggests that this region determines the subcellular localization of individual Rab proteins. To confirm this hypothesis, we constructed Rab35-Rab10 chimera proteins by exchanging their C-terminal domains with one another. Confocal microscopy of RAW264 cells expressing EGFP-fused Rab35-Rab10 chimeras has indicated that the C-terminal region of Rab35 is critical for its plasma membrane localization. Furthermore, we were able to determine that a basic amino acid cluster exists in the C-terminal region of Rab35 and that Rab35 localization shifts to the Golgi membrane when the number of basic amino acids in this region is reduced. Thus, it is likely that the approximate 30-amino acid C-terminal region containing basic clusters is responsible for Rab35 plasma membrane localization and that its preferential localization depends on the number of basic amino acids.

Keywords: C-terminus; Rab35; basic clusters; confocal microscopy; plasma membrane.

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Conflict of interest statement

IVThe authors declare that there are no conflicts of interest.

Figures

Fig. 1.
Fig. 1.
Comparison between the amino acid sequences of Rab35 and its subfamily members. (A) Phylogenetic tree of the Rab35 subfamily. The phylogenetic tree was drawn using DNA sequences from the National Center for Biotechnology Information (NCBI) RefSeq database. (B) The multiple sequence alignments of mouse Rab35, Rab1a, Rab8a, and Rab10 were created using the Clustal X ver. 2.1 program developed by the European Bioinformatics Institute. In the clustal consensus line (c.c.), an asterisk (*) indicates the positions which have a fully conserved residue, a colon (:) indicates conservation between groups of amino acids with strongly similar properties, and a period (.) indicates conservation between groups of amino acids with weakly similar properties. Conserved and semi-conserved amino acids are shaded light gray. (C) Amino acid similarity values between Rab35 and Rab10 were obtained using Clustal X ver. 2.1.
Fig. 2.
Fig. 2.
The subcellular localizations of Rab35, Rab10, and Rab35–Rab10 chimeras in live RAW264 cells. (A) The schematic diagram showing chimera mutants created by exchanging the C-terminal hypervariable domain (HVD) of Rab35 (blue) and Rab10 (green). (B) Confocal microscopy showing the localization of Rab35, Rab10, Rab35/10, and Rab10/35 chimeras in live RAW264 cells. Cells were transfected using a pEGFP-fused Rab protein and observed using a Zeiss LSM700 inverted microscope. An image at one time point from time-lapse microscopy is presented. EGFP-Rab35 was observed on the plasma membrane. EGFP-Rab10 was predominantly observed in the perinuclear Golgi region. EGFP-Rab35/10 chimera exhibited a similar localization to Rab10. EGFP-Rab10/35 chimera was localized to the plasma membrane similar to Rab35. Bars = 10 μm.
Fig. 3.
Fig. 3.
Changes in localization of Rab35 by reducing the number of basic amino acids at the C-terminus. (A) The C-terminal amino acid sequences of Rab35-wt, Rab35-2E, and Rab35-4E. The HVD of wild-type Rab35 has a characteristic basic cluster (red letters). In Rab35-2E and -4E, the basic amino acids lysine (K) and arginine (R) were replaced with glutamate (E), an acidic amino acid. The substituted glutamate residues in Rab35-2E and -4E are shown in blue. (B) Wild-type EGFP-Rab35, point-mutated EGFP-Rab35-2E, and EGFP-Rab35-4E were transfected into RAW264 cells and observed using a confocal laser microscope. Although wild-type Rab35 was localized to the plasma membrane, Rab35-2E was found to be localized in some organelles in the cytoplasm. Notably, Rab35-4E was exclusively accumulated in the perinuclear region. (C) RAW264 cells expressing point-mutated Rab35 (Rab35-2E, Rab-4E) were immunostained with anti-GM130, a cis-Golgi marker. By reducing the number of basic amino acids at the C-terminus, Rab35 mutants were accumulated primarily in the Golgi apparatus. Bars = 10 μm.
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