Systematic Screens for Proteins That Interact with the Mucolipidosis Type IV Protein TRPML1

Abstract

Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.

Transient Receptor Potential Channels as Drug Targets: From the Science of Basic Research to the Art of Medicine

“…TRPML1 also associates with members of the lysosome-associated protein transmembrane family, which is important for the organelle remodeling (Spooner et al, 2013). TRPML1 might be also coupled to transducer of regulated CREB activity-1, which supports a role in organelle fusion (Venkatachalam et al, 2013).…”

The role of TRPMLs in endolysosomal trafficking and function

“…Indeed, unbiased protein interaction studies have found that TRPML1 physically interacts with several mitochondrial proteins [127]. Therefore, lysosomal dysfunction in cells lacking TRPML1 lead to concomitant aberrations in mitochondrial function, morphology, Ca 2+ buffering capacity, and caspase-mediated cell death [128].…”

“…A large-scale screen identified several candidate proteins that interact with TRPML1 [127]. Briefly, some of the interesting interactors of TRPML1 in this screen involved the cytoskeleton modifying small G-proteins such as Rac2 and Cdc42 (indicating a potential role of TRPML1 in cytoskeleton regulation), lysosomal enzymes such as Cathepsin B, members of the solute carrier family of transporters, the ER Ca 2+ release channel IP 3 -receptor as well as the Ca 2+ transporting ATPase (SERCA2), markers of the ER-Golgi intermediate compartments (ERGIC) and Yif1, which regulate ER to Golgi trafficking of proteins (these interactions may mediate the exit of TRPML1 and associated proteins from the ER), and peroxisome-associated Pex16 [127].…”

“…Briefly, some of the interesting interactors of TRPML1 in this screen involved the cytoskeleton modifying small G-proteins such as Rac2 and Cdc42 (indicating a potential role of TRPML1 in cytoskeleton regulation), lysosomal enzymes such as Cathepsin B, members of the solute carrier family of transporters, the ER Ca 2+ release channel IP 3 -receptor as well as the Ca 2+ transporting ATPase (SERCA2), markers of the ER-Golgi intermediate compartments (ERGIC) and Yif1, which regulate ER to Golgi trafficking of proteins (these interactions may mediate the exit of TRPML1 and associated proteins from the ER), and peroxisome-associated Pex16 [127]. Interestingly, both approaches identified several mitochondrial proteins as TRPML1-interactors that among others include NADH dehydrogenase, ATP synthase subunits, and subunits of the mitochondrial voltage-dependent ion channel (VDAC, also known as Porin) [127]. …”

A small molecule restores function to TRPML1 mutant isoforms responsible for mucolipidosis type IV

“…In recent years, TRPML1 has been characterized using electrophysiological tools and its basic cation channel properties have been investigated [9][10][11][12][13][14][15][16][17][18][19] , its protein-protein interaction network has been explored [20][21][22][23][24] , and knockout mouse models have been generated and investigated [25][26][27][28] . However, effective treatment options for MLIV patients are still missing.…”