Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Abstract

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the transGolgi network (TGN). Previous studies on Shiga toxinsuggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes-TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factorregulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069-7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.

DNAJC13 mutations in Parkinson disease

“…Preliminary functional analysis indicates that the expression of DNAJC13 p.Asn855Ser in COS7 cells leads to accumulation of TF in endosomal compartments, suggesting interference with endosomal compartmentalization or trafficking as the pathological mechanism of disease. The importance of DNAJC13 function in endosomal trafficking is evident from several studies showing alterations in trafficking of well-known retromer cargos when its function is perturbed (13,20). The function of retromer itself is linked to PD through the observation that mutations in another retromer subunit, VPS35, can cause familial parkinsonism (6,7).…”

“…It is interesting to note that mutations in DNAJC6 have been recently identified in juvenile parkinsonism (18), and genome-wide association studies have identified susceptibility alleles for PD at chromosome 4p16 spanning the GAK/ DGKQ locus (19). Interestingly, DNAJC13 has also been shown to interact with the retromer component sorting nexin 1 (20). The retromer generates clathrin-independent carriers that mediate retrograde targeting of selective cargo from endosomes to the trans-Golgi network (21).…”

“…The retromer generates clathrin-independent carriers that mediate retrograde targeting of selective cargo from endosomes to the trans-Golgi network (21). DNAJC13 is thus thought to function by coordinating retromer with endosomal clathrin coats, either by regulating clathrin dynamics to allow for the sorting of cargo from clathrin subdomains into the retrograde vesicles or by creating clathrin-free patches on endosomal membranes allowing for sites of retromer-mediated vesicle formation (12,20). Preliminary functional analysis indicates that the expression of DNAJC13 p.Asn855Ser in COS7 cells leads to accumulation of TF in endosomal compartments, suggesting interference with endosomal compartmentalization or trafficking as the pathological mechanism of disease.…”

How Ricin and Shiga Toxin Reach the Cytosol of Target Cells: Retrotranslocation from the Endoplasmic Reticulum

“…Furthermore, the clathrin associated Hcs70 cochaperone RME-8 regulates endosomal trafficking of STx (Popoff et al 2009) whilst having no effect on the transport of cholera toxin (Girard et al 2005). …”

Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics

“…In addition to binding Hsc70, RME-8 has also been shown to associate and co-localize with the endosome membrane remodeling component SNX1 (10,18). SNX1 when complexed with SNX2 recruits the Vps26-Vps29-Vps35 retromer trimer to form the heteropentameric coat, also known as retromer (19).…”