Melanosome transport defects in melan-ln cells may be rescued by the introduction of exogenous Mlph. (A–F) A time course of recovery of melanosome transport dynamics after exogenous expression of Mlph. Coverslip grown melan-ln melanocytes were coinjected with pEGFP-Mlph and Texas red dextran and melanosome distribution in injected cells was observed using phase-contrast optics. (A) The distribution of Texas red dextran and arrows indicate injected cells; (B–F) the position of melanosomes in the injected cells at the indicated time in minutes after injection. Panels G–I show that recovery of melanosome transport defects in melan-ln cells is sensitive to the level of expression of Mlph. Cells were transfected with plasmid encoding 6xmyc-Mlph, fixed 48 h later, stained using anti-myc antibodies, and observed by confocal microscopy. (G) A representative image of the distribution of overexpressed 6xmyc-Mlph in a number of melan-ln cells expressing varying levels of exogenous protein; (H) the distribution of melanosomes observed using transmission optics. White traces in G and H indicate that perimeter of transfected cells. (I) The relationship between Mlph expression level (average fluorescence intensity/pixel/cell) in a typical population of transfected cells and the level of rescue of melanosome transport defects in each cell (as described in Materials and Methods). ■ and □, cells expressing wild-type Mlph or nonfunctional mutant Mlph, respectively. Bar, 20 μm.

EFBD but not GTBD is important for Mlph mediated recruitment of MyoVa to melanosomal membranes. Melan-ln cells were transfected with plasmids encoding Mlph molecules in which the MBD is specifically disrupted, fixed 48 h later, and stained with antibodies that detect myc-tagged Mlph and MyoVa. (A, E, I, M, and Q) The distribution of the indicated overexpressed myc-tagged Mlph proteins. (B, F, J, N, and R) The distribution of endogenous MyoVa. (C, G, K, O, and S) Merged fluorescence images showing the extent of overlap myc-tagged Mlph (green) and MyoVa (red). (D, H, L, P, and T) The corresponding transmission images showing the distribution of pigment in transfected cells. Arrows indicate the position of myc-Mlph associated with pigmented melanosomes. Bar, 20 μm.

Mutations of Mlph that disrupt the integrity of the 440-483 coiled-coil disrupt Mlph function. (A) Prediction of coiled-coil structure in wild-type Mlph and mutant constructs correlates with rescue of melan-ln melanosome transport defects. The amino acid sequence of wild-type and mutant Mlph molecules was analyzed using the MultiCoils coiled-coil prediction algorithm available online at http://multicoil.lcs.mit.edu/cgi-bin/multicoil. The chart shows in detail the analyses of aa 440-492 of each construct as determined using the MultiCoils prediction method (results from the COILS method available at http://www.ch.embnet.org/software/COILS_form.html show similar trends; unpublished data). For the chimeric construct MlphCC coil analysis of aa 455-507 shown as ClustalW alignment of protein sequences indicate that this region of the chimeric constructs corresponds to 440-492 of wild-type Mlph. Solid lines indicate constructs that allow rescue of melan-ln melanosome transport defects, and broken lines indicate constructs that fail to do so. (B–U) melan-ln melanocytes were transfected with plasmids encoding the indicated mutant Mlph molecules. Cells were fixed 48 h later and stained with antibodies that detect overexpressed Mlph and MyoVa. Panels B, F, J, N, and R show the distribution of overexpressed Mlph; panels C, G, K, O, and S show the distribution of endogenous MyoVa; panels D, H, L, P, and T are merged images showing the extent of colocalization of myc-Mlph (green) with MyoVa (red); and panels E, I, M, Q, and U are the corresponding transmission images showing the distribution of melanosomes. Arrows indicate colocalization of overexpressed protein with pigmented melanosomes. Bar, 20 μm.

Quantification of the extent of rescue of melanosome transport by different Mlph mutant constructs. Melan-ln cells were transfected with the indicated constructs, and the rescue efficiency for each cell was determined as described in Materials and Methods. (A, B, and C) Representative results of individual experiments for constructs transfected in Figures 4, 5, and 6, respectively, together with Mlph and Slp1 controls. In all cases n = 50. Boxes indicate 25th/75th percentile, bars within boxes indicate median values, outer bars indicate 5th/95th percentile, and outer points indicate outliers.

Summary of the functional characteristic of Mlph constructs used in this study. The top part of the figure is a schematic representation of the domain structure of Mlph showing the regions previously shown to be involved in interaction with other proteins. In the bottom part of the figure solid lines indicate wild-type Mlph sequence, dashed lines indicate internal truncations, gray shaded areas indicate non-Mlph sequence within chimeric proteins, and letters A and X indicate the position of ala or other substitutions in point mutants. Each construct is scored + or − for its ability to be recruited to melanosomes, the ability to recruit MyoVa to melanosome and its ability to rescue melanosome transport defects in melan-ln cells.

Both N-terminus SHD regions but not the ZnF are essential for melanosome association of Mlph. Melan-ln melanocytes were transfected with plasmids encoding myc-tagged fragments of Mlph, fixed 48 h later, and stained with antibodies (as described in Materials and Methods). (A) A schematic representation of the subdomains of the N-terminus R27BD of Mlph. (B, D, F, H, J, L, N, P, and S) The distribution of the indicated overexpressed protein. (C, E, G, I, K, M, O, R, and T) Transmission images showing the distribution of melanosomes. (Q) The distribution of filamentous actin as detected using Texas-red phalloidin. Arrows indicate areas of colocalization of overexpressed protein with pigmented melanosomes (B, C, L, and M) or filamentous actin (P and Q). Bar, 20 μm.

Rescue of melan-ln melanosome transport defects requires part of the ABD in addition to R27BD and MBD sequences. Melan-ln melanocytes were transfected with plasmids encoding the indicated C-terminus–truncated Mlph molecules, fixed 48 h later, and stained with antibodies to detect myc-tagged Mlph and MyoVa. (A, E, I, M, Q, and U) The distribution of myc-tagged Mlph molecule as indicated. (B, F, J, N, R, and V) The distribution of endogenous MyoVa. (C, G, K, O, S, and W) Merged fluorescence images showing the overlap of myc-tagged Mlph (green) and MyoVa (red). (D, H, L, P, T, and X) The corresponding transmission images showing the distribution of pigment in transfected cells. Arrows indicate colocalization of overexpressed protein with pigmented melanosomes. Bar, 20 μm.

Measurement of the interaction of truncated Mlph proteins with MyoVa. (A and B) In vitro interaction of truncated Mlph proteins with the cargo binding C-terminus tail of MyoVa. One hundred picomoles of the indicated GST-Mlph protein was incubated with 100 pmol of MBP-MyoVaMSGTA in the presence of glutathione-Sepharose beads as described in Materials and Methods. Bound proteins were precipitated, eluted from the beads, and subjected to immunoblotting using α-GST and α-MBP antibodies. (C) Assay of interaction of truncated Mlph with full-length, endogenous MyoVa. melan-ln cells infected with adenovirus expressing V5-Mlph, lysed, and subjected to immunoprecipitation with anti-V5 antibodies. Immune complexes were then probed with anti-V5 and anti-MyoVa antibodies. For both assays bands (see autoradiographs in A and C) were quantified using Aida software and relative binding (see bar chart) was calculated by dividing the band intensity of MyoVa by the band intensity of Mlph. Experiments were carried out in triplicate. The migration of molecular-weight standards is indicated on the left of each blot.