Identification and characterization of Iporin as a novel interaction partner for rab1

BACKGROUND: The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. RESULTS: Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R) as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD). Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. CONCLUSION: Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus

LRRK2 transport is regulated by its novel interacting partner Rab32

Leucine-rich repeat kinase 2 (LRRK2) is a multi-domain 280 kDa protein that is linked to Parkinson's disease (PD). Mutations especially in the GTPase and kinase domains of LRRK2 are the most common causes of heritable PD and are also found in sporadic forms of PD. Although the cellular function of LRRK2 is largely unknown there is increasing evidence that these mutations cause cell death due to autophagic dysfunction and mitochondrial damage. Here, we demonstrate a novel mechanism of LRRK2 binding and transport, which involves the small GTPases Rab32 and Rab38. Rab32 and its closest homologue Rab38 are known to organize the trans-Golgi network and transport of key enzymes in melanogenesis, whereas their function in non-melanogenic cells is still not well understood. Cellular processes such as autophagy, mitochondrial dynamics, phagocytosis or inflammatory processes in the brain have previously been linked to Rab32. Here, we demonstrate that Rab32 and Rab38, but no other GTPase tested, directly interact with LRRK2. GFP-Trap analyses confirmed the interaction of Rab32 with the endogenous LRRK2. In yeast two-hybrid experiments we identified a predicted coiled-coil motif containing region within the aminoterminus of LRRK2 as the possible interacting domain. Fluorescence microscopy demonstrated a co-localization of Rab32 and LRRK2 at recycling endosomes and transport vesicles, while overexpression of a constitutively active mutant of Rab32 led to an increased co-localization with Rab7/9 positive perinuclear late endosomes/MVBs. Subcellular fractionation experiments supported the novel role of Rab32 in LRRK2 late endosomal transport and sorting in the cell. Thus, Rab32 may regulate the physiological functions of LRRK2

Rab32 interacts with SNX6 and affects retromer-dependent Golgi trafficking

The Rab family of small GTPases regulate various aspects of cellular dynamics in eukaryotic cells. Membrane trafficking has emerged as central to the functions of leucine-rich repeat kinase 2 (LRRK2), which is associated with inherited and sporadic forms of Parkinson’s disease (PD). Rabs act as both regulators of the catalytic activity and targets for serine/threonine phosphorylation by LRRK2. Rab32, Rab38 and Rab29 have been shown to regulate LRRK2 sub-cellular localization through direct interactions. Recently, Rab29 was shown to escort LRRK2 to the Golgi apparatus and activate the phosphorylation of Rab8 and Rab10. Rab32 is linked to multiple cellular functions including endosomal trafficking, mitochondrial dynamics, and melanosome biogenesis. A missense mutation in Rab32 has also recently been linked to PD. Here, we demonstrate that Rab32 directly interacts with sorting nexin 6 (SNX6). SNX6 is a transient subunit of the retromer, an endosome-Golgi retrieval complex whose Vps35 subunit is strongly associated with PD. We could further show that localization of cation-independent mannose-6-phosphate receptors, which are recycled to the trans-Golgi network (TGN) by the retromer, was affected by both Rab32 and SNX6. These data imply that Rab32 is linked to SNX6/retromer trafficking at the Golgi, and also suggests a possible connection between the retromer and Rab32 in the trafficking and biological functions of LRRK2

A New Mint1 Isoform, but Not the Conventional Mint1, Interacts with the Small GTPase Rab6

Small GTPases of the Rab family are important regulators of a large variety of different cellular functions such as membrane organization and vesicle trafficking. They have been shown to play a role in several human diseases. One prominent member, Rab6, is thought to be involved in the development of Alzheimer’s Disease, the most prevalent mental disorder worldwide. Previous studies have shown that Rab6 impairs the processing of the amyloid precursor protein (APP), which is cleaved to β-amyloid in brains of patients suffering from Alzheimer’s Disease. Additionally, all three members of the Mint adaptor family are implied to participate in the amyloidogenic pathway. Here, we report the identification of a new Mint1 isoform in a yeast two-hybrid screening, Mint1 826, which lacks an eleven amino acid (aa) sequence in the conserved C-terminal region. Mint1 826, but not the conventional Mint1, interacts with Rab6 via the PTB domain. This interaction is nucleotide-dependent, Rab6-specific and influences the subcellular localization of Mint1 826. We were able to detect and sequence a corresponding proteolytic peptide derived from cellular Mint1 826 by mass spectrometry proving the absence of aa 495–505 and could show that the deletion does not influence the ability of this adaptor protein to interact with APP. Taking into account that APP interacts and co-localizes with Mint1 826 and is transported in Rab6 positive vesicles, our data suggest that Mint1 826 bridges APP to the small GTPase at distinct cellular sorting points, establishing Mint1 826 as an important player in regulation of APP trafficking and processing

Stress response of fire salamander larvae differs between habitat types

Schulte L, Oswald P, Mühlenhaupt M, et al. Stress response of fire salamander larvae differs between habitat types. Royal Society Open Science. 2024;11(4).The larvae of the European fire salamander (Salamandra salamandra) can inhabit two different habitats: streams and ponds. Streams are characterized by lower predation risks and higher food availability. Thus, ponds are considered a less suitable habitat. To investigate the differential impacts of these two habitats on larval physiology, we measured the stress response of larvae. After successfully validating the measure of water-borne corticosterone release rates in fire salamander larvae, we measured the baseline and stress-induced corticosterone of 64 larvae from ponds and streams in the field. We found that larvae in ponds have a higher baseline and stress-induced corticosterone levels. Additionally, we performed a reciprocal transplant experiment (RTE) and tested whether larvae can adapt their stress responses to changing habitats. After two weeks, we did not find an increase in corticosterone levels when comparing stress-induced corticosterone values with baseline corticosterone values in larvae transferred into ponds, irrespective of their habitat of origin. However, larvae transferred into streams still exhibited an increase in the stress-induced corticosterone response in comparison with the baseline values. These results show that non-invasive hormone measurements can provide information on the habitat quality and potential adaptation and thus emphasize the potential for its use in conservation efforts

Stress response of fire salamander larvae differs between habitat types

Schulte L, Oswald P, Mühlenhaupt M, et al. Stress response of fire salamander larvae differs between habitat types. Bielefeld University; 2023

Water-borne hormone extraction and determination of corticosterone - tables from Stress response of fire salamander larvae differs between habitat types

Reference table

LRRK2-GFP subcellular localization in DsRed-Monomer-Rab32 wt and DsRed-Monomer-Rab32 Q85L overexpressing NIH3T3 cells.

<p>(A) Graphical representation of the most common subcellular features of LRRK2-GFP in DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L expressing cells. 1: pericentriolar endosome; 2: transport vesicles; 3: perinuclear aggregates. (B–D) Microscopic analysis of NIH3T3 cells co-transfected with plasmids encoding DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L and LRRK2-GFP. In every cell the LRRK2-GFP channel was analyzed for the occurrence of the features transport vesicles, pericentriolar endosomes and perinuclear aggregates. (-) LRRK2-GFP alone: n = 71 cells from 5 independent experiments; LRRK2-GFP and DsRed-Monomer-Rab32 wildtype: n = 15 cells from 3 independent experiments, LRRK2-GFP and DsRed-Monomer-Rab32 Q85L: n = 21 cells from 5 independent experiments.</p

Co-localization analysis of Rab32 wt and the constitutively active mutant Rab32 Q85L with different endosomal markers.

<p>(A) NIH3T3 cells were co-transfected with plasmids encoding the recycling endosome marker GFP-Rab11B and DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L, fixed and analyzed by fluorescence microscopy. Scale bar = 10 µm. (B) Cells were transfected with plasmids encoding for GFP-Rab32 wt or GFP-Rab32 Q85L followed by fixation and subsequent immunofluorescence staining of Rab7. Scale bar = 10 µm. (C–E) NIH3T3 cells expressing either GFP-Rab32 wt or GFP-Rab32 Q85L were fixed and stained for Rab7. (C) Microscopic analysis of GFP-Rab32 Q85L that co-localized with endogenous Rab7 (arrows) in the perinuclear area. Non co-localizing Rab7 was indicated by arrowheads. The perinuclear area was defined by the red (outlines nucleus) and the yellow line (outer border for perinuclear area). The image illustrates the cellular area used for the following analysis. (D) Rab7 perinuclear aggregates co-localizing with GFP-Rab32 wt or GFP-Rab32 Q85L and non co-localizing ones. ctrl. = control (untransfected IHKE-1 cells) (E) Quantification of perinuclear Rab7-positive structures. ctrl. (control): perinuclear Rab7 in untransfected cells; wt/Q85L: perinuclear Rab7 co-localizing with GFP-Rab32 constructs. control/Rab32 wt/Rab32 Q85L: n = 1660/18/167 structures in 67/18/60 cells, 1/1/3 independent experiments. Statistical significance was tested by a Student's T-test: p_control-wt = 0.02; p_control-Q85L<0.005; p_wt-Q85L = 0.02. Scale bar = 10 µm.</p